Compounds and uses thereof

ABSTRACT

The present disclosure features compounds useful for the treatment of BAF complex-related disorders.

BACKGROUND

The invention relates to compounds useful for modulating BRG1- orBRM-associated factors (BAF) complexes. In particular, the inventionrelates to compounds useful for treatment of disorders associated withBAF complex function.

Chromatin regulation is essential for gene expression, and ATP-dependentchromatin remodeling is a mechanism by which such gene expressionoccurs. The human Switch/Sucrose Non-Fermentable (SWI/SNF) chromatinremodeling complex, also known as BAF complex, has two SWI2-like ATPasesknown as BRG1 (Brahma-related gene-1) and BRM (Brahma). Thetranscription activator BRG1, also known as ATP-dependent chromatinremodeler SMARCA4, is encoded by the SMARCA4 gene on chromosome 19. BRG1is overexpressed in some cancer tumors and is needed for cancer cellproliferation. BRM, also known as probable global transcriptionactivator SNF2L2 and/or ATP-dependent chromatin remodeler SMARCA2, isencoded by the SMARCA2 gene on chromosome 9 and has been shown to beessential for tumor cell growth in cells characterized by loss of BRG1function mutations. Deactivation of BRG and/or BRM results in downstreameffects in cells, including cell cycle arrest and tumor suppression.

SUMMARY

The present invention features compounds useful for modulating a BAFcomplex. In some embodiments, the compounds are useful for the treatmentof disorders associated with an alteration in a BAF complex, e.g., adisorder associated with an alteration in one or both of the BRG1 andBRM proteins. The compounds of the invention, alone or in combinationwith other pharmaceutically active agents, can be used for treating suchdisorders.

In an aspect, the invention features a compound,N-(1-((4-(6-(2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-methoxy-1-oxopropan-2-yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide,or a pharmaceutically acceptable salt thereof, having the structure:

In some embodiments, the compound, or a pharmaceutically acceptable saltthereof, has the structure:

In some embodiments, the compound, or a pharmaceutically acceptable saltthereof, has the structure:

In some embodiments, the compound, or a pharmaceutically acceptable saltthereof, has the structure:

In another aspect, the invention features a compound,N-(1-((4-(6-(2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-(methoxy-d3)-1-oxopropan-2-yl-3,3-d2)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide,or a pharmaceutically acceptable salt thereof, having the structure:

In some embodiments, the compound, or a pharmaceutically acceptable saltthereof, has the structure:

In some embodiments, the compound, or a pharmaceutically acceptable saltthereof, has the structure:

In some embodiments, the compound, or a pharmaceutically acceptable saltthereof, has the structure:

In another aspect, the invention features a pharmaceutical compositionincluding any of the foregoing compounds and a pharmaceuticallyacceptable excipcient.

In another aspect, the invention features a method of decreasing theactivity of a BAF complex in a cell or subject. This method includescontacting the cell with, or administering to the subject, an effectiveamount of any of the foregoing compounds or pharmaceutical compositions.

In another aspect, the invention features a method of inhibiting BRM ina cell or subject. This method includes contacting the cell with, oradministering to the subject, an effective amount of any of theforegoing compounds or pharmaceutical compositions.

In another aspect, the invention features a method of inhibiting BRG1 ina cell or subject. This method includes contacting the cell with, oradministering to the subject, an effective amount of any of theforegoing compounds or pharmaceutical compositions.

In another aspect, the invention features a method of inhibiting BRM andBRG1 in a cell or subject. This method includes contacting the cellwith, or administering to the subject, an effective amount of any of theforegoing compounds or pharmaceutical compositions. In another aspect,the invention features a method of inducing apoptosis in a cell orsubject. This method includes contacting the cell with, or administeringto the subject, an effective amount of any of the foregoing compounds orpharmaceutical compositions.

In some embodiments of any of the foregoing methods, the cell is acancer cell and/or the subject has cancer. In another aspect, theinvention features a method of treating a BAF complex-related disorderin a subject in need thereof. This method includes administering to thesubject an effective amount of any of the foregoing compounds orpharmaceutical compositions.

In another aspect, the invention features a method of treating adisorder related to a BRG1 loss of function mutation in a subject inneed thereof. This method includes administering to the subject aneffective amount of any of the foregoing compounds or pharmaceuticalcompositions. In some embodiments, the subject is determined to have aBRG1 loss of function disorder (e.g., the disorder and/or subject hasbeen determined to include cells with a loss of BRG1 function mutation).

In some embodiments of the foregoing methods, the BAF complex-relateddisorder or disorder related to a BRG1 loss of function mutation is acancer, a viral infection, Coffin Siris, Neurofibromatosis (e.g., NF-1,NF-2, or Schwannomatosis), or Multiple Meningioma.

In another aspect, the invention features a method of treating cancer ina subject in need thereof. This method includes administering to thesubject an effective amount of any of the foregoing compounds orpharmaceutical compositions.

In another aspect, the invention features a method of reducing tumorgrowth of cancer in a subject in need thereof in need thereof. Thismethod includes administering to the subject an effective amount of anyof the foregoing compounds or pharmaceutical compositions.

In another aspect, the invention features a method of suppressingmetastatic progression of cancer in a subject in need thereof. Thismethod includes administering an effective amount of any of theforegoing compounds or pharmaceutical compositions.

In another aspect, the invention features a method of suppressingmetastatic colonization (e.g., metastatic colonization to the lungand/or brain) of cancer in a subject in need thereof. This methodincludes administering an effective amount of any of the foregoingcompounds or pharmaceutical compositions.

In another aspect, the invention features a method of reducing the leveland/or activity of BRG1 and/or BRM in a cancer in a cell or subject inneed thereof. This method includes contacting the cell with, oradministering to the subject, an effective amount of any of theforegoing compounds or pharmaceutical compositions.

In some embodiments of any of the foregoing methods, the cancer isnon-small cell lung cancer, colorectal cancer, bladder cancer, cancer ofunknown primary, glioma, breast cancer, melanoma, non-melanoma skincancer, endometrial cancer, esophagogastric cancer, esophageal cancer,pancreatic cancer, hepatobiliary cancer, soft tissue sarcoma, ovariancancer, head and neck cancer, renal cell carcinoma, bone cancer,non-Hodgkin lymphoma, small-cell lung cancer, prostate cancer, embryonaltumor, germ cell tumor, cervical cancer, thyroid cancer, salivary glandcancer, gastrointestinal neuroendocrine tumor, uterine sarcoma,gastrointestinal stromal tumor, CNS cancer, thymic tumor, Adrenocorticalcarcinoma, appendiceal cancer, small bowel cancer, penile cancer, bonecancer, or a hematologic cancer. In some embodiments of any of theforegoing methods, the cancer is esophageal cancer.

In some embodiments of any of the foregoing methods, the cancer isnon-small cell lung cancer, colorectal cancer, bladder cancer, cancer ofunknown primary, glioma, breast cancer, melanoma, non-melanoma skincancer, endometrial cancer, penile cancer, bone cancer, renal cellcarcinoma, prostate cancer, or a hematologic cancer. In some embodimentsof any of the foregoing methods, the cancer is non-small cell lungcancer.

In some embodiments of any of the foregoing methods, the cancer ismelanoma, prostate cancer, breast cancer, bone cancer, renal cellcarcinoma, or a hematologic cancer.

In some embodiments, the cancer is melanoma (e.g., uveal melanoma,mucosal melanoma, or cutaneous melanoma). In some embodiments, thecancer is prostate cancer. In some embodiments, the cancer is ahematologic cancer (e.g., multiple myeloma, large cell lymphoma, acuteT-cell leukemia, acute myeloid leukemia, myelodysplastic syndrome,immunoglobulin A lambda myeloma, diffuse mixed histiocytic andlymphocytic lymphoma, B-cell lymphoma, acute lymphoblastic leukemia(e.g., T-cell acute lymphoblastic leukemia or B-cell acute lymphoblasticleukemia), diffuse large cell lymphoma, or non-Hodgkin's lymphoma). Insome embodiments, the cancer is breast cancer (e.g., an ER positivebreast cancer, an ER negative breast cancer, triple positive breastcancer, or triple negative breast cancer). In some embodiments, thecancer is a bone cancer (e.g., Ewing's sarcoma). In some embodiments,the cancer is a renal cell carcinoma (e.g., a MicrophthalmiaTranscription Factor (MITF) family translocation renal cell carcinoma(tRCC)).

In some embodiments, the cancer expresses BRG1 and/or BRM protein and/orthe cell or subject has been identified as expressing BRG1 and/or BRM.In some embodiments, the cancer expresses

BRG1 protein and/or the cell or subject has been identified asexpressing BRG1. In some embodiments, the cancer expresses BRM proteinand/or the cell or subject has been identified as expressing BRM. Insome embodiments, the subject or cancer has and/or has been identifiedas having a BRG1 loss of function mutation. In some embodiments, thesubject or cancer has and/or has been identified as having a BRM loss offunction mutation.

In some embodiments of any of the foregoing methods, the cancer has orhas been determined to have one or more BRG1 mutations (e.g., homozygousmutations). In some embodiments, the one or more BRG1 mutations includesa mutation in the ATPase catalytic domain of the protein. In someembodiments, the one or more BRG1 mutations include a deletion at theC-terminus of BRG1.

In some embodiments of any of the foregoing methods, the cancer does nothave, or has been determined not to have, an epidermal growth factorreceptor (EGFR) mutation. In some embodiments of any of the foregoingmethods, the cancer does not have, or has been determined not to have,an anaplastic lymphoma kinase (ALK) driver mutation. In some embodimentsof any of the foregoing methods, the cancer has, or has been determinedto have, a KRAS mutation.

In some embodiments the cancer has, or has been determined to have, amutation in GNAQ. In some embodiments the cancer has, or has beendetermined to have, a mutation in GNA11. In some embodiments the cancerhas, or has been determined to have, a mutation in PLCB4. In someembodiments the cancer has, or has been determined to have, a mutationin CYSLTR2. In some embodiments the cancer has, or has been determinedto have, a mutation in BAP1. In some embodiments the cancer has, or hasbeen determined to have, a mutation in SF3B1. In some embodiments thecancer has, or has been determined to have, a mutation in EIF1AX. Insome embodiments the cancer has, or has been determined to have, a TFE3translocation. In some embodiments the cancer has, or has beendetermined to have, a TFEB translocation. In some embodiments the cancerhas, or has been determined to have, a MITF translocation. In someembodiments the cancer has, or has been determined to have, an EZH2mutation. In some embodiments the cancer has, or has been determined tohave, a SUZ12 mutation. In some embodiments the cancer has, or has beendetermined to have, an EED mutation.

In some embodiments, the cancer is metastatic. For example, the cancerincludes cells exhibiting migration and/or invasion of migrating cellsand/or includes cells exhibiting endothelial recruitment and/orangiogenesis. The metastatic cancer may be spread via seeding thesurface of the peritoneal, pleural, pericardial, or subarachnoid spaces.Alternatively, the metastatic cancer may be spread via the lymphaticsystem, or spread hematogenously. In some embodiments, the cancer is acell migration cancer (e.g., a non-metastatic cell migration cancer).

In some embodiments of any of the foregoing methods, the cancer is drugresistant (e.g., the cancer has been determined to be resistant, orlikely to be resistant, to chemotherapeutic or cytotoxic agents such asby genetic markers, or is likely to be resistant, to chemotherapeutic orcytotoxic agents such as a cancer that has failed to respond to achemotherapeutic or cytotoxic agent) and/or has failed to respond to aprior therapy (e.g., a chemotherapeutic or cytotoxic agent,immunotherapy, surgery, radiotherapy, thermotherapy, orphotocoagulation, or a combination thereof).

In some embodiments, the cancer is resistant to and/or has failed torespond to vemurafenib, dacarbazine, a CTLA4 inhibitor, a PD1 inhibitor,interferon therapy, a BRAF inhibitor, a MEK inhibitor, radiotherapy,temozolimide, irinotecan, a CAR-T therapy, herceptin, perjeta,tamoxifen, xeloda, docetaxol, platinum agents such as carboplatin,taxanes such as paclitaxel and docetaxel, ALK inhibitors, METinihibitors, alimta, abraxane, doxorubicin, gemcitabine, avastin,halaven, neratinib, a PARP inhibitor, brilanestrant, an mTOR inhibitor,topotecan, gemzar, a VEGFR2 inhibitor, a folate receptor antagonist,demcizumab, fosbretabulin, or a PDL1 inhibitor, or combinations thereof.

In some embodiments of any of the foregoing methods, the cancer isresistant to and/or has failed to respond to dacarbazine, temozolomide,cisplatin, treosulfan, fotemustine, IMCgp100, a CTLA-4 inhibitor (e.g.,ipilimumab), a PD-1 inhibitor (e.g., nivolumab or pembrolizumab), aPD-L1 inhibitor (e.g., atezolizumab, avelumab, or durvalumab), amitogen-activated protein kinase (MEK) inhibitor (e.g., selumetinib,binimetinib, or tametinib), and/or a protein kinase C (PKC) inhibitor(e.g., sotrastaurin or IDE196).

In some embodiments of any of the foregoing methods, the cancer isresistant to and/or failed to respond to a previously administeredtherapeutic used for the treatment of uveal melanoma, e.g., a MEKinhibitor or PKC inhibitor. For example, in some embodiments, the canceris resistant to and/or failed to respond to a mitogen-activated proteinkinase (MEK) inhibitor (e.g., selumetinib, binimetinib, or tametinib),and/or a protein kinase C (PKC) inhibitor (e.g., sotrastaurin orIDE196).

In some embodiments, the method further includes administering to thesubject or contacting the cell with an anticancer therapy, e.g., achemotherapeutic or cytotoxic agent, immunotherapy, surgery,radiotherapy, thermotherapy, or photocoagulation, or combinationsthereof. In some embodiments, the anticancer therapy is achemotherapeutic or cytotoxic agent, e.g., an antimetabolite,antimitotic, antitumor antibiotic, asparagine-specific enzyme,bisphosphonates, antineoplastic, alkylating agent, DNA-Repair enzymeinhibitor, histone deacetylase inhibitor, corticosteroid, demethylatingagent, immunomodulatory, janus-associated kinase inhibitor,phosphinositide 3-kinase inhibitor, proteasome inhibitor, or tyrosinekinase inhibitor, or combinations thereof.

In some embodiments, the compound of the invention is used incombination with another anti-cancer therapy used for the treatment ofuveal melanoma such as surgery, a MEK inhibitor, and/or a PKC inhibitor,or combinations thereof. For example, in some embodiments, the methodfurther comprises performing surgery prior to, subsequent to, or at thesame time as administration of the compound of the invention. In someembodiments, the method further comprises administration of a MEKinhibitor (e.g., selumetinib, binimetinib, or tametinib) and/or a PKCinhibitor (e.g., sotrastaurin or IDE196) prior to, subsequent to, or atthe same time as administration of the compound of the invention.

In some embodiments, the anticancer therapy and the compound of theinvention are administered within 28 days (e.g., within 21 days, within14 days, or within 7 days) of each other and each in an amount thattogether are effective to treat the subject.

In another aspect, the disclosure provides a method for treating a viralinfection in a subject in need thereof. This method includesadministering to the subject an effective amount of any of the foregoingcompounds, or pharmaceutical compositions. In some embodiments, theviral infection is an infection with a virus of the Retroviridae familysuch as the lentiviruses (e.g., Human immunodeficiency virus (HIV)) anddeltaretroviruses (e.g., human T cell leukemia virus I (HTLV-I), human Tcell leukemia virus II (HTLV-II)), a virus of the Hepadnaviridae family(e.g., hepatitis B virus (HBV)), a virus of the Flaviviridae family(e.g., hepatitis C virus (HCV)), a virus of the Adenoviridae family(e.g., Human Adenovirus), a virus of the Herpesviridae family (e.g.,Human cytomegalovirus (HCMV), Epstein-Barr virus, herpes simplex virus 1(HSV-1), herpes simplex virus 2 (HSV-2), human herpesvirus 6 (HHV-6),

Herpesvitus K*, CMV, varicella-zoster virus), a virus of thePapillomaviridae family (e.g., Human Papillomavirus (HPV, HPV El)), avirus of the Parvoviridae family (e.g., Parvovirus B19), a virus of thePolyomaviridae family (e.g., JC virus and BK virus), a virus of theParamyxoviridae family (e.g., Measles virus), or a virus of theTogaviridae family (e.g., Rubella virus). In some embodiments of any ofthe foregoing methods, the effective amount of the compound reduces thelevel and/or activity of BRG1 by at least 5% (e.g., at least 6%, atleast 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or at least 99%) as compared to a reference.

In some embodiments of any of the foregoing methods, the effectiveamount of the compound reduces the level and/or activity of BRG1 by atleast 5% (e.g., at least 6%, at least 7%, at least 8%, at least 9%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99%) as compared to areference for at least 12 hours (e.g., at least 14 hours, at least 16hours, at least 18 hours, at least 20 hours, at least 22 hours, at least24 hours, at least 30 hours, at least 36 hours, at least 48 hours, atleast 72 hours, at least 4 days, at least 5 days, at least 6 days, atleast 7 days, at least 14 days, at least 21 days, at least 28 days, ormore).

In some embodiments of any of the foregoing methods, the effectiveamount of the compound reduces the level and/or activity of BRM by atleast 5% (e.g., at least 6%, at least 7%, at least 8%, at least 9%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99%) as compared to areference.

In some embodiments of any of the foregoing methods, the effectiveamount of the compound reduces the level and/or activity of BRM by atleast 5% (e.g., at least 6%, at least 7%, at least 8%, at least 9%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99%) as compared to areference for at least 12 hours (e.g., at least 14 hours, at least 16hours, at least 18 hours, at least 20 hours, at least 22 hours, at least24 hours, at least 30 hours, at least 36 hours, at least 48 hours, atleast 72 hours, at least 4 days, at least 5 days, at least 6 days, atleast 7 days, at least 14 days, at least 21 days, at least 28 days, ormore).

In some embodiments, the effective amount of the compound of theinvention is an amount effective to inhibit metastatic colonization ofthe cancer to the liver and/or brain.

Chemical Terms

Compounds of the invention can have one or more asymmetric carbon atomsand can exist in the form of optically pure enantiomers, mixtures ofenantiomers such as, for example, racemates, optically purediastereoisomers, mixtures of diastereoisomers, diastereoisomericracemates, or mixtures of diastereoisomeric racemates. The opticallyactive forms can be obtained for example by resolution of the racemates,by asymmetric synthesis or asymmetric chromatography (chromatographywith a chiral adsorbents or eluant). That is, certain of the disclosedcompounds may exist in various stereoisomeric forms. Stereoisomers arecompounds that differ only in their spatial arrangement. Enantiomers arepairs of stereoisomers whose mirror images are not superimposable, mostcommonly because they contain an asymmetrically substituted carbon atomthat acts as a chiral center. “Enantiomer” means one of a pair ofmolecules that are mirror images of each other and are notsuperimposable. Diastereomers are stereoisomers that are not related asmirror images, most commonly because they contain two or moreasymmetrically substituted carbon atoms and represent the configurationof substituents around one or more chiral carbon atoms. Enantiomers of acompound can be prepared, for example, by separating an enantiomer froma racemate using one or more well-known techniques and methods, such as,for example, chiral chromatography and separation methods based thereon.The appropriate technique and/or method for separating an enantiomer ofa compound described herein from a racemic mixture can be readilydetermined by those of skill in the art. “Racemate” or “racemic mixture”means a compound containing two enantiomers, wherein such mixturesexhibit no optical activity; i.e., they do not rotate the plane ofpolarized light. “Geometric isomer” means isomers that differ in theorientation of substituent atoms in relationship to a carbon-carbondouble bond, to a cycloalkyl ring, or to a bridged bicyclic system.Atoms (other than H) on each side of a carbon—carbon double bond may bein an E (substituents are on opposite sides of the carbon—carbon doublebond) or Z (substituents are oriented on the same side) configuration.“R,” “S,” “S*,” “R*,” “E,” “Z,” “cis,” and “trans,” indicateconfigurations relative to the core molecule. Certain of the disclosedcompounds may exist in atropisomeric forms. Atropisomers arestereoisomers resulting from hindered rotation about single bonds wherethe steric strain barrier to rotation is high enough to allow for theisolation of the conformers. The compounds of the invention may beprepared as individual isomers by either isomer-specific synthesis orresolved from an isomeric mixture. Conventional resolution techniquesinclude forming the salt of a free base of each isomer of an isomericpair using an optically active acid (followed by fractionalcrystallization and regeneration of the free base), forming the salt ofthe acid form of each isomer of an isomeric pair using an opticallyactive amine (followed by fractional crystallization and regeneration ofthe free acid), forming an ester or amide of each of the isomers of anisomeric pair using an optically pure acid, amine or alcohol (followedby chromatographic separation and removal of the chiral auxiliary), orresolving an isomeric mixture of either a starting material or a finalproduct using various well known chromatographic methods. When thestereochemistry of a disclosed compound is named or depicted bystructure, the named or depicted stereoisomer is at least 60%, 70%, 80%,90%, 99%, or 99.9% by weight relative to the other stereoisomers. When asingle enantiomer is named or depicted by structure, the depicted ornamed enantiomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weightoptically pure. When a single diastereomer is named or depicted bystructure, the depicted or named diastereomer is at least 60%, 70%, 80%,90%, 99%, or 99.9% by weight pure. Percent optical purity is the ratioof the weight of the enantiomer or over the weight of the enantiomerplus the weight of its optical isomer. Diastereomeric purity by weightis the ratio of the weight of one diastereomer or over the weight of allthe diastereomers. When the stereochemistry of a disclosed compound isnamed or depicted by structure, the named or depicted stereoisomer is atleast 60%, 70%, 80%, 90%, 99%, or 99.9% by mole fraction pure relativeto the other stereoisomers. When a single enantiomer is named ordepicted by structure, the depicted or named enantiomer is at least 60%,70%, 80%, 90%, 99%, or 99.9% by mole fraction pure. When a singlediastereomer is named or depicted by structure, the depicted or nameddiastereomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by molefraction pure. Percent purity by mole fraction is the ratio of the molesof the enantiomer or over the moles of the enantiomer plus the moles ofits optical isomer. Similarly, percent purity by moles fraction is theratio of the moles of the diastereomer or over the moles of thediastereomer plus the moles of its isomer. When a disclosed compound isnamed or depicted by structure without indicating the stereochemistry,and the compound has at least one chiral center, it is to be understoodthat the name or structure encompasses either enantiomer of the compoundfree from the corresponding optical isomer, a racemic mixture of thecompound, or mixtures enriched in one enantiomer relative to itscorresponding optical isomer. When a disclosed compound is named ordepicted by structure without indicating the stereochemistry and has twoor more chiral centers, it is to be understood that the name orstructure encompasses a diastereomer free of other diastereomers, anumber of diastereomers free from other diastereomeric pairs, mixturesof diastereomers, mixtures of diastereomeric pairs, mixtures ofdiastereomers in which one diastereomer is enriched relative to theother diastereomer(s), or mixtures of diastereomers in which one or morediastereomer is enriched relative to the other diastereomers. Theinvention embraces all of these forms.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds that differ only in the presence of one or moreisotopically enriched atoms. Exemplary isotopes that can be incorporatedinto compounds of the present invention include isotopes of hydrogen,carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, andiodine, such as ²H_(,) ³H_(,) ¹¹C_(,) ¹³C_(,) ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O,¹⁸O, ³²P, ³³P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I and ¹²⁵I. Isotopically-labeledcompounds (e.g., those labeled with ³H and ¹⁴C)) can be useful incompound or substrate tissue distribution assays. Tritiated (i.e., ³H)and carbon-14 (i.e., ¹⁴C)) isotopes can be useful for their ease ofpreparation and detectability. Further, substitution with heavierisotopes such as deuterium (i.e., ²H) may afford certain therapeuticadvantages resulting from greater metabolic stability (e.g., increasedin vivo half-life or reduced dosage requirements). In some embodiments,one or more hydrogen atoms are replaced by ²H or ³H, or one or morecarbon atoms are replaced by ¹³C- or ¹⁴C-enriched carbon. Positronemitting isotopes such as ¹⁵O, ¹³N, ¹¹C, and ¹⁸F are useful for positronemission tomography (PET) studies to examine substrate receptoroccupancy. Preparations of isotopically labelled compounds are known tothose of skill in the art. For example, isotopically labeled compoundscan generally be prepared by following procedures analogous to thosedisclosed for compounds of the present invention described herein, bysubstituting an isotopically labeled reagent for a non-isotopicallylabeled reagent.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present disclosure; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Definitions

In this application, unless otherwise clear from context, (i) the term“a” may be understood to mean “at least one”; (ii) the term “or” may beunderstood to mean “and/or”; and (iii) the terms “comprising” and“including” may be understood to encompass itemized components or stepswhether presented by themselves or together with one or more additionalcomponents or steps.

As used herein, the terms “about” and “approximately” refer to a valuethat is within 10% above or below the value being described. Forexample, the term “about 5 nM” indicates a range of from 4.5 to 5.5 nM.As used herein, the term “administration” refers to the administrationof a composition (e.g., a compound or a preparation that includes acompound as described herein) to a subject or system. Administration toan animal subject (e.g., to a human) may be by any appropriate route.For example, in some embodiments, administration may be bronchial(including by bronchial instillation), buccal, enteral, interdermal,intra-arterial, intradermal, intragastric, intramedullary,intramuscular, intranasal, intraperitoneal, intrathecal, intratumoral,intravenous, intraventricular, mucosal, nasal, oral, rectal,subcutaneous, sublingual, topical, tracheal (including by intratrachealinstillation), transdermal, vaginal, and vitreal.

As used herein, the term “BAF complex” refers to the BRG1- orHBRM-associated factors complex in a human cell.

As used herein, the term “BAF complex-related disorder” refers to adisorder that is caused or affected by the level of activity of a BAFcomplex. As used herein, the term “BRG1 loss of function mutation”refers to a mutation in BRG1 that leads to the protein having diminishedactivity (e.g., at least 1% reduction in BRG1 activity, for example 2%,5%, 10%, 25%, 50%, or 100% reduction in BRG1 activity). Exemplary BRG1loss of function mutations include, but are not limited to, a homozygousBRG1 mutation and a deletion at the C-terminus of BRG1.

As used herein, the term “BRG1 loss of function disorder” refers to adisorder (e.g., cancer) that exhibits a reduction in BRG1 activity(e.g., at least 1% reduction in BRG1 activity, for example 2%, 5%, 10%,25%, 50%, or 100% reduction in BRG1 activity).

The term “cancer” refers to a condition caused by the proliferation ofmalignant neoplastic cells, such as tumors, neoplasms, carcinomas,sarcomas, leukemias, and lymphomas.

As used herein, a “combination therapy” or “administered in combination”means that two (or more) different agents or treatments are administeredto a subject as part of a defined treatment regimen for a particulardisease or condition. The treatment regimen defines the doses andperiodicity of administration of each agent such that the effects of theseparate agents on the subject overlap. In some embodiments, thedelivery of the two or more agents is simultaneous or concurrent and theagents may be co-formulated. In some embodiments, the two or more agentsare not co-formulated and are administered in a sequential manner aspart of a prescribed regimen. In some embodiments, administration of twoor more agents or treatments in combination is such that the reductionin a symptom, or other parameter related to the disorder is greater thanwhat would be observed with one agent or treatment delivered alone or inthe absence of the other. The effect of the two treatments can bepartially additive, wholly additive, or greater than additive (e.g.,synergistic). Sequential or substantially simultaneous administration ofeach therapeutic agent can be effected by any appropriate routeincluding, but not limited to, oral routes, intravenous routes,intramuscular routes, and direct absorption through mucous membranetissues. The therapeutic agents can be administered by the same route orby different routes. For example, a first therapeutic agent of thecombination may be administered by intravenous injection while a secondtherapeutic agent of the combination may be administered orally.

The term “CTLA-4 inhibitor,” as used herein, refers to a compound suchas an antibody capable of inhibiting the activity of the protein that inhumans is encoded by the CTLA4 gene. Known CTLA-4 inhibitors includeipilimumab.

By “determining the level” of a protein or RNA is meant the detection ofa protein or an RNA, by methods known in the art, either directly orindirectly. “Directly determining” means performing a process (e.g.,performing an assay or test on a sample or “analyzing a sample” as thatterm is defined herein) to obtain the physical entity or value.“Indirectly determining” refers to receiving the physical entity orvalue from another party or source (e.g., a third party laboratory thatdirectly acquired the physical entity or value). Methods to measureprotein level generally include, but are not limited to, westernblotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, surfaceplasmon resonance, chemiluminescence, fluorescent polarization,phosphorescence, immunohistochemical analysis, matrix-assisted laserdesorption/ionization time-of-flight (MALDI-TOF) mass spectrometry,liquid chromatography (LC)-mass spectrometry, microcytometry,microscopy, fluorescence activated cell sorting (FACS), and flowcytometry, as well as assays based on a property of a protein including,but not limited to, enzymatic activity or interaction with other proteinpartners. Methods to measure RNA levels are known in the art andinclude, but are not limited to, quantitative polymerase chain reaction(qPCR) and Northern blot analyses.

By a “decreased level” or an “increased level” of a protein or RNA ismeant a decrease or increase, respectively, in a protein or RNA level,as compared to a reference (e.g., a decrease or an increase by about 5%,about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about150%, about 200%, about 300%, about 400%, about 500%, or more; adecrease or an increase of more than about 10%, about 15%, about 20%,about 50%, about 75%, about 100%, or about 200%, as compared to areference; a decrease or an increase by less than about 0.01-fold, about0.02-fold, about 0.1-fold, about 0.3-fold, about 0.5-fold, about0.8-fold, or less; or an increase by more than about 1.2-fold, about1.4-fold, about 1.5-fold, about 1.8-fold, about 2.0-fold, about3.0-fold, about 3.5-fold, about 4.5-fold, about 5.0-fold, about 10-fold,about 15-fold, about 20-fold, about 30-fold, about 40-fold, about50-fold, about 100-fold, about 1000-fold, or more). A level of a proteinmay be expressed in mass/vol (e.g., g/dL, mg/mL, μg/mL, ng/mL) orpercentage relative to total protein in a sample.

By “decreasing the activity of a BAF complex” is meant decreasing thelevel of an activity related to a BAF complex, or a related downstreameffect. A non-limiting example of decreasing an activity of a BAFcomplex is Sox2 activation. The activity level of a BAF complex may bemeasured using any method known in the art, e.g., the methods describedin Kadoch et al. Cell, 2013, 153, 71-85, the methods of which are hereinincorporated by reference.

As used herein, the term “derivative” refers to naturally-occurring,synthetic, and semi-synthetic analogues of a compound, peptide, protein,or other substance described herein. A derivative of a compound,peptide, protein, or other substance described herein may retain orimprove upon the biological activity of the original material.

A cancer “determined to be drug resistant,” as used herein, refers to acancer that is drug resistant, based on unresponsiveness or decreasedresponsiveness to a chemotherapeutic agent, or is predicted to be drugresistant based on a prognostic assay (e.g., a gene expression assay).

By a “drug resistant” is meant a cancer that does not respond, orexhibits a decreased response to, one or more chemotherapeutic agents(e.g., any agent described herein).

As used herein, the term “failed to respond to a prior therapy” or“refractory to a prior therapy,” refers to a cancer that progresseddespite treatment with the therapy.

As used herein, the term “inhibiting BRM” and/or “inhibiting BRG1”refers to blocking or reducing the level or activity of the ATPasecatalytic binding domain or the bromodomain of the protein. BRM and/orBRG1 inhibition may be determined using methods known in the art, e.g.,a BRM and/or BRG1 ATPase assay, a Nano DSF assay, or a BRM and/or BRG1Luciferase cell assay.

As used herein, the term “LXS196,” also known as IDE196, refers to thePKC inhibitor having the structure:

or a pharmaceutically acceptable salt thereof.

As used herein, “metastatic nodule” refers to an aggregation of tumorcells in the body at a site other than the site of the original tumor.

As used herein, “metastatic cancer” refers to a tumor or cancer in whichthe cancer cells forming the tumor have a high potential to or havebegun to, metastasize, or spread from one location to another locationor locations within a subject, via the lymphatic system or viahaematogenous spread, for example, creating secondary tumors within thesubject. Such metastatic behavior may be indicative of malignant tumors.In some cases, metastatic behavior may be associated with an increase incell migration and/or invasion behavior of the tumor cells.

Examples of cancers that can be defined as metastatic include but arenot limited to lung cancer (e.g., non-small cell lung cancer), breastcancer, ovarian cancer, colorectal cancer, biliary tract cancer, bladdercancer, brain cancer including glioblastomas and medullablastomas,cervical cancer, choriocarcinoma, endometrial cancer, esophageal cancer,gastric cancer, hematological neoplasms, multiple myeloma, leukemia,intraepithelial neoplasms, liver cancer, lymphomas, neuroblastomas, oralcancer, pancreatic cancer, prostate cancer, sarcoma, skin cancerincluding melanoma, basocellular cancer, squamous cell cancer,testicular cancer, stromal tumors, germ cell tumors, thyroid cancer, andrenal cancer.

“Non-metastatic cell migration cancer” as used herein refers to cancersthat do not migrate via the lymphatic system or via haematogenousspread.

The term “PD-1 inhibitor,” as used herein, refers to a compound such asan antibody capable of inhibiting the activity of the protein that inhumans is encoded by the PDCD1 gene. Known PD-1 inhibitors includenivolumab, pembrolizumab, pidilizumab, BMS 936559, and atezolizumab.

The term “PD-L1 inhibitor,” as used herein, refers to a compound such asan antibody capable of inhibiting the activity of the protein that inhumans is encoded by the CD274 gene. Known PD-L1 inhibitors includeatezolizumab and durvalumab.

The term “pharmaceutical composition,” as used herein, represents acomposition containing a compound described herein formulated with apharmaceutically acceptable excipient and appropriate for administrationto a mammal, for example a human. Typically, a pharmaceuticalcomposition is manufactured or sold with the approval of a governmentalregulatory agency as part of a therapeutic regimen for the treatment ofdisease in a mammal. Pharmaceutical compositions can be formulated, forexample, for oral administration in unit dosage form (e.g., a tablet,capsule, caplet, gelcap, or syrup); for topical administration (e.g., asa cream, gel, lotion, or ointment); for intravenous administration(e.g., as a sterile solution free of particulate emboli and in a solventsystem suitable for intravenous use); or in any other pharmaceuticallyacceptable formulation.

A “pharmaceutically acceptable excipient,” as used herein, refers to anyingredient other than the compounds described herein (for example, avehicle capable of suspending or dissolving the active compound) andhaving the properties of being substantially nontoxic andnon-inflammatory in a patient. Excipients may include, for example:antiadherents, antioxidants, binders, coatings, compression aids,disintegrants, dyes (colors), emollients, emulsifiers, fillers(diluents), film formers or coatings, flavors, fragrances, glidants(flow enhancers), lubricants, preservatives, printing inks, sorbents,suspensing or dispersing agents, sweeteners, and waters of hydration.

As used herein, the term “pharmaceutically acceptable salt” means anypharmaceutically acceptable salt of a compound described herein.Pharmaceutically acceptable salts of any of the compounds describedherein may include those that are within the scope of sound medicaljudgment, suitable for use in contact with the tissues of humans andanimals without undue toxicity, irritation, allergic response and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example,pharmaceutically acceptable salts are described in: Berge et al., J.Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts:Properties, Selection, and Use, (Eds. P. H. Stahl and C. G. Wermuth),Wiley-VCH, 2008. The salts can be prepared in situ during the finalisolation and purification of the compounds described herein orseparately by reacting a free base group with a suitable organic acid.

The compounds of the invention may have ionizable groups so as to becapable of preparation as pharmaceutically acceptable salts. These saltsmay be, e.g., acid addition salts involving inorganic or organic acidsor the salts may, in the case of acidic forms of the compounds of theinvention be prepared from inorganic or organic bases. Frequently, thecompounds are prepared or used as pharmaceutically acceptable saltsprepared as addition products of pharmaceutically acceptable acids orbases. Suitable pharmaceutically acceptable acids and bases and methodsfor preparation of the appropriate salts are well-known in the art.Salts may be prepared from pharmaceutically acceptable non-toxic acidsand bases including inorganic and organic acids and bases.

“Progression-free survival” as used herein, refers to the length of timeduring and after medication or treatment during which the disease beingtreated (e.g., cancer) does not get worse.

“Proliferation” as used in this application involves reproduction ormultiplication of similar forms (cells) due to constituting (cellular)elements.

By a “reference” is meant any useful reference used to compare proteinor RNA levels. The reference can be any sample, standard, standardcurve, or level that is used for comparison purposes. The reference canbe a normal reference sample or a reference standard or level. A“reference sample” can be, for example, a control, e.g., a predeterminednegative control value such as a “normal control” or a prior sampletaken from the same subject; a sample from a normal healthy subject,such as a normal cell or normal tissue; a sample (e.g., a cell ortissue) from a subject not having a disease; a sample from a subjectthat is diagnosed with a disease, but not yet treated with a compound ofthe invention; a sample from a subject that has been treated by acompound of the invention; or a sample of a purified protein or

RNA (e.g., any described herein) at a known normal concentration. By“reference standard or level” is meant a value or number derived from areference sample. A “normal control value” is a pre-determined valueindicative of non-disease state, e.g., a value expected in a healthycontrol subject. Typically, a normal control value is expressed as arange (“between X and Y”), a high threshold (“no higher than X”), or alow threshold (“no lower than X”). A subject having a measured valuewithin the normal control value for a particular biomarker is typicallyreferred to as “within normal limits” for that biomarker. A normalreference standard or level can be a value or number derived from anormal subject not having a disease or disorder (e.g., cancer); asubject that has been treated with a compound of the invention. Inpreferred embodiments, the reference sample, standard, or level ismatched to the sample subject sample by at least one of the followingcriteria: age, weight, sex, disease stage, and overall health. Astandard curve of levels of a purified protein or RNA, e.g., anydescribed herein, within the normal reference range can also be used asa reference.

As used herein, “slowing the spread of metastasis” refers to reducing orstopping the formation of new loci; or reducing, stopping, or reversingthe tumor load.

As used herein, the term “subject” refers to any organism to which acomposition in accordance with the invention may be administered, e.g.,for experimental, diagnostic, prophylactic, and/or therapeutic purposes.Typical subjects include any animal (e.g., mammals such as mice, rats,rabbits, non-human primates, and humans). A subject may seek or be inneed of treatment, require treatment, be receiving treatment, bereceiving treatment in the future, or be a human or animal who is undercare by a trained professional for a particular disease or condition.

As used herein, the terms “treat,” “treated,” or “treating” meantherapeutic treatment or any measures whose object is to slow down(lessen) an undesired physiological condition, disorder, or disease, orobtain beneficial or desired clinical results. Beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms; diminishment of the extent of a condition, disorder, ordisease; stabilized (i.e., not worsening) state of condition, disorder,or disease; delay in onset or slowing of condition, disorder, or diseaseprogression; amelioration of the condition, disorder, or disease stateor remission (whether partial or total); an amelioration of at least onemeasurable physical parameter, not necessarily discernible by thepatient; or enhancement or improvement of condition, disorder, ordisease. Treatment includes eliciting a clinically significant responsewithout excessive levels of side effects. Treatment also includesprolonging survival as compared to expected survival if not receivingtreatment. Compounds of the invention may also be used to“prophylactically treat” or “prevent” a disorder, for example, in asubject at increased risk of developing the disorder.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present disclosure; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating inhibition of cell proliferation ofseveral cancer cell lines by a BRG1/BRM inhibitor (Compound A).

FIG. 2 is a graph illustrating inhibition of cell proliferation of uvealmelanoma cell line 92-1 by a

BRG1/BRM inhibitor (Compound A), a MEK inhibitor (Selumetinib), and aPKC inhibitor (LXS196).

FIG. 3 is a graph illustrating inhibition of cell proliferation of uvealmelanoma cell line MP41 by a BRG1/BRM inhibitor (Compound A), a MEKinhibitor (Selumetinib), and a PKC inhibitor (LXS196).

FIG. 4 is a graph illustrating inhibition of cell proliferation ofseveral cancer cell lines by a BRG1/BRM inhibitor (Compound B).

FIG. 5 is a graph illustrating the area under the curves (AUCs)calculated from dose-response curves for cancer cell lines treated witha BRG1/BRM inhibitor (Compound B).

FIG. 6 is a graph illustrating inhibition of cell proliferation of uvealmelanoma and non-small cell lung cancer cell lines by a BRG1/BRMinhibitor (Compound B).

FIG. 7 is a graph illustrating inhibition of cell proliferation of uvealmelanoma cell line 92-1 by a BRG1/BRM inhibitor (Compound B), a MEKinhibitor (Selumetinib), and a PKC inhibitor (LXS196).

FIG. 8 is a graph illustrating inhibition of cell proliferation of uvealmelanoma cell line MP41 by a BRG1/BRM inhibitor (Compound B), a MEKinhibitor (Selumetinib), and a PKC inhibitor (LXS196).

FIG. 9 is a graph illustrating inhibition of cell proliferation ofparental and PKC-inhibitor refractory uveal melanoma cell lines by a PKCinhibitor (LXS196).

FIG. 10 is a graph illustrating inhibition of cell proliferation ofparental and PKC-inhibitor refractory uveal melanoma cell lines by aBRG1/BRM inhibitor (Compound B).

FIG. 11 is a graph illustrating inhibition of tumor growth in miceengrafted with uveal melanoma cell lines by a BRG1/BRM inhibitor(Compound C).

FIG. 12 is an illustration of the size of tumors from mice engraftedwith uveal melanoma cell lines and dosed with a BRG1/BRM inhibitor(Compound C).

FIG. 13 is a graph illustrating body weight change of mice engraftedwith uveal melanoma cell lines and dosed with a BRG1/BRM inhibitor(Compound C).

FIG. 14 is a graph illustrating inhibition of cell proliferation ofseveral uveal melanoma cell lines byN-((S)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-methoxy-1-oxopropan-2-yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide.

FIG. 15 is a graph illustrating inhibition of tumor growth in miceengrafted with uveal melanoma cell lines byN-((S)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-methoxy-1-oxopropan-2-yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide.

FIG. 16 is a graph illustrating body weight change of mice engraftedwith uveal melanoma cell lines and dosed withN-((S)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-methoxy-1-oxopropan-2-yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide.

DETAILED DESCRIPTION

The present disclosure features compounds useful for the inhibition ofBRG1 and/or BRM. These compounds may be used to modulate the activity ofa BAF complex, for example, for the treatment of a

BAF-related disorder, such as cancer. Exemplary compounds,orpharmaceutically acceptable salts thereof, described herein includecompounds having the structure:

Other embodiments, as well as exemplary methods for the synthesis ofproduction of these compounds, are described herein.

Pharmaceutical Uses

The compounds described herein are useful in the methods of theinvention and, while not bound by theory, are believed to exert theirability to modulate the level, status, and/or activity of a BAF complex,i.e., by inhibiting the activity of the BRG1 and/or BRM proteins withinthe BAF complex in a mammal.

BAF complex-related disorders include, but are not limited to, BRG1 lossof function mutation-related disorders.

An aspect of the present invention relates to methods of treatingdisorders related to BRG1 loss of function mutations such as cancer(e.g., non-small cell lung cancer, colorectal cancer, bladder cancer,cancer of unknown primary, glioma, breast cancer, melanoma, non-melanomaskin cancer, endometrial cancer, or penile cancer) in a subject in needthereof. In some embodiments, the present invention relates to methodsof treating melanoma (e.g., uveal melanoma), prostate cancer, breastcancer, bone cancer, renal cell carcinoma, or a hematologic cancer.

In some embodiments, the compound is administered in an amount and for atime effective to result in one or more (e.g., two or more, three ormore, four or more) of: (a) reduced tumor size, (b) reduced rate oftumor growth, (c) increased tumor cell death (d) reduced tumorprogression, (e) reduced number of metastases, (f) reduced rate ofmetastasis, (g) decreased tumor recurrence (h) increased survival ofsubject, (i) increased progression free survival of subject.

Treating cancer can result in a reduction in size or volume of a tumor.For example, after treatment, tumor size is reduced by 5% or greater(e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater) relativeto its size prior to treatment. Size of a tumor may be measured by anyreproducible means of measurement. For example, the size of a tumor maybe measured as a diameter of the tumor.

Treating cancer may further result in a decrease in number of tumors.For example, after treatment, tumor number is reduced by 5% or greater(e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater) relativeto number prior to treatment. Number of tumors may be measured by anyreproducible means of measurement, e.g., the number of tumors may bemeasured by counting tumors visible to the naked eye or at a specifiedmagnification (e.g., 2×, 3×, 4×, 5×, 10×, or 50×).

Treating cancer can result in a decrease in number of metastatic nodulesin other tissues or organs distant from the primary tumor site. Forexample, after treatment, the number of metastatic nodules is reduced by5% or greater (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% orgreater) relative to number prior to treatment. The number of metastaticnodules may be measured by any reproducible means of measurement. Forexample, the number of metastatic nodules may be measured by countingmetastatic nodules visible to the naked eye or at a specifiedmagnification (e.g., 2×, 10×, or 50×).

Treating cancer can result in an increase in average survival time of apopulation of subjects treated according to the present invention incomparison to a population of untreated subjects. For example, theaverage survival time is increased by more than 30 days (more than 60days, 90 days, or 120 days). An increase in average survival time of apopulation may be measured by any reproducible means. An increase inaverage survival time of a population may be measured, for example, bycalculating fora population the average length of survival followinginitiation of treatment with the compound of the invention. An increasein average survival time of a population may also be measured, forexample, by calculating for a population the average length of survivalfollowing completion of a first round of treatment with apharmaceutically acceptable salt of the invention.

Treating cancer can also result in a decrease in the mortality rate of apopulation of treated subjects in comparison to an untreated population.For example, the mortality rate is decreased by more than 2% (e.g., morethan 5%, 10%, or 25%). A decrease in the mortality rate of a populationof treated subjects may be measured by any reproducible means, forexample, by calculating for a population the average number ofdisease-related deaths per unit time following initiation of treatmentwith a pharmaceutically acceptable salt of the invention. A decrease inthe mortality rate of a population may also be measured, for example, bycalculating for a population the average number of disease-relateddeaths per unit time following completion of a first round of treatmentwith a pharmaceutically acceptable salt of the invention.

Exemplary cancers that may be treated by the invention include, but arenot limited to, non-small cell lung cancer, small-cell lung cancer,colorectal cancer, bladder cancer, glioma, breast cancer, melanoma,non-melanoma skin cancer, endometrial cancer, esophagogastric cancer,esophageal cancer, pancreatic cancer, hepatobiliary cancer, soft tissuesarcoma, ovarian cancer, head and neck cancer, renal cell carcinoma,bone cancer, non-Hodgkin lymphoma, prostate cancer, embryonal tumor,germ cell tumor, cervical cancer, thyroid cancer, salivary gland cancer,gastrointestinal neuroendocrine tumor, uterine sarcoma, gastrointestinalstromal tumor, CNS cancer, thymic tumor, Adrenocortical carcinoma,appendiceal cancer, small bowel cancer, hematologic cancer, and penilecancer.

Combination Formulations and Uses Thereof

The compounds of the invention can be combined with one or moretherapeutic agents. In particular, the therapeutic agent can be one thattreats or prophylactically treats any cancer described herein.

Combination Therapies

A compound of the invention can be used alone or in combination with anadditional therapeutic agent, e.g., other agents that treat cancer orsymptoms associated therewith, or in combination with other types oftreatment to treat cancer. In combination treatments, the dosages of oneor more of the therapeutic compounds may be reduced from standarddosages when administered alone. For example, doses may be determinedempirically from drug combinations and permutations or may be deduced byisobolographic analysis (e.g., Black et al., Neurology 65:S3-S6, 2005).In this case, dosages of the compounds when combined should provide atherapeutic effect.

In some embodiments, the second therapeutic agent is a chemotherapeuticagent (e.g., a cytotoxic agent or other chemical compound useful in thetreatment of cancer). These include alkylating agents, antimetabolites,folic acid analogs, pyrimidine analogs, purine analogs and relatedinhibitors, vinca alkaloids, epipodopyyllotoxins, antibiotics,L-Asparaginase, topoisomerase inhibitors, interferons, platinumcoordination complexes, anthracenedione substituted urea, methylhydrazine derivatives, adrenocortical suppressant,adrenocorticosteroides, progestins, estrogens, antiestrogen, androgens,antiandrogen, and gonadotropin-releasing hormone analog. Also includedis 5-fluorouracil (5-FU), leucovorin (LV), irenotecan, oxaliplatin,capecitabine, paclitaxel and doxetaxel. Non-limiting examples ofchemotherapeutic agents include alkylating agents such as thiotepa andcyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammaII and calicheamicinomegall (see, e.g., Agnew, Chem. Intl. Ed Engl. 33:183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Adriamycin®(doxorubicin, including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5- FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;

aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., Taxol®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABraxane®,cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTaxotere® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;

Gemzar gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum coordination complexes such as cisplatin, oxaliplatin andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine; Navelbine® vinorelbine; novantrone;teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate;irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Two or more chemotherapeutic agents canbe used in a cocktail to be administered in combination with the firsttherapeutic agent described herein. Suitable dosing regimens ofcombination chemotherapies are known in the art and described in, forexample, Saltz et al. (1999) Proc ASCO 18:233a and Douillard et al.(2000) Lancet 355:1041-7.

In some embodiments, the second therapeutic agent is a therapeutic agentwhich is a biologic such a cytokine (e.g., interferon or an interleukin(e.g., IL-2)) used in cancer treatment. In some embodiments the biologicis an anti-angiogenic agent, such as an anti-VEGF agent, e.g.,bevacizumab (Avastin®). In some embodiments the biologic is animmunoglobulin-based biologic, e.g., a monoclonal antibody (e.g., ahumanized antibody, a fully human antibody, an Fc fusion protein or afunctional fragment thereof) that agonizes a target to stimulate ananti-cancer response, or antagonizes an antigen important for cancer.Such agents include Rituxan (Rituximab); Zenapax (Daclizumab); Simulect(Basiliximab); Synagis (Palivizumab); Remicade (Infliximab); Herceptin(Trastuzumab); Mylotarg (Gemtuzumab ozogamicin); Campath (Alemtuzumab);Zevalin (Ibritumomab tiuxetan); Humira (Adalimumab); Xolair(Omalizumab); Bexxar (Tositumomab-I-131); Raptiva (Efalizumab); Erbitux(Cetuximab); Avastin (Bevacizumab); Tysabri (Natalizumab); Actemra(Tocilizumab); Vectibix (Panitumumab); Lucentis (Ranibizumab); Soliris(Eculizumab); Cimzia (Certolizumab pegol); Simponi (Golimumab); Ilaris(Canakinumab); Stelara (Ustekinumab); Arzerra (Ofatumumab); Prolia(Denosumab); Numax (Motavizumab); ABThrax (Raxibacumab); Benlysta(Belimumab); Yervoy (Ipilimumab); Adcetris (Brentuximab Vedotin);Perjeta (Pertuzumab); Kadcyla (Ado-trastuzumab emtansine); and Gazyva(Obinutuzumab). Also included are antibody-drug conjugates.

The second agent may be a therapeutic agent which is a non-drugtreatment. For example, the second therapeutic agent is radiationtherapy, cryotherapy, hyperthermia and/or surgical excision of tumortissue.

The second agent may be a checkpoint inhibitor. In one embodiment, theinhibitor of checkpoint is an inhibitory antibody (e.g., a monospecificantibody such as a monoclonal antibody). The antibody may be, e.g.,humanized or fully human. In some embodiments, the inhibitor ofcheckpoint is a fusion protein, e.g., an Fc-receptor fusion protein. Insome embodiments, the inhibitor of checkpoint is an agent, such as anantibody, that interacts with a checkpoint protein. In some embodiments,the inhibitor of checkpoint is an agent, such as an antibody, thatinteracts with the ligand of a checkpoint protein. In some embodiments,the inhibitor of checkpoint is an inhibitor (e.g., an inhibitoryantibody or small molecule inhibitor) of CTLA-4 (e.g., an anti-CTLA4antibody such as ipilimumab/Yervoy or tremelimumab). In someembodiments, the inhibitor of checkpoint is an inhibitor (e.g., aninhibitory antibody or small molecule inhibitor) of PD-1 (e.g.,nivolumab/Opdivo®; pembrolizumab/Keytruda®; pidilizumab/CT-011). In someembodiments, the inhibitor of checkpoint is an inhibitor (e.g., aninhibitory antibody or small molecule inhibitor) of PDL1 (e.g.,MPDL3280A/RG7446; MED14736; MSB0010718C; BMS 936559). In someembodiments, the inhibitor of checkpoint is an inhibitor (e.g., aninhibitory antibody or Fc fusion or small molecule inhibitor) of PDL2(e.g., a PDL2/lg fusion protein such as AMP 224). In some embodiments,the inhibitor of checkpoint is an inhibitor (e.g., an inhibitoryantibody or small molecule inhibitor) of B7-H3 (e.g., MGA271), B7-H4,BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1,CHK2, A2aR, B-7 family ligands, or a combination thereof.

In some embodiments, the compound of the invention is used incombination with another anti-cancer therapy used for the treatment ofuveal melanoma such as surgery, a MEK inhibitor, and/or a PKC inhibitor,or a combination thereof. For example, in some embodiments, the methodfurther comprises performing surgery prior to, subsequent to, or at thesame time as administration of the compound of the invention. In someembodiments, the method further comprises administration of a MEKinhibitor (e.g., selumetinib, binimetinib, or tametinib) and/or a PKCinhibitor (e.g., sotrastaurin or IDE196) prior to, subsequent to, or atthe same time as administration of the compound of the invention.

In any of the combination embodiments described herein, the first andsecond therapeutic agents are administered simultaneously orsequentially, in either order. The first therapeutic agent may beadministered immediately, up to 1 hour, up to 2 hours, up to 3 hours, upto 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to, 8 hours,up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13hours, 14 hours, up to hours 16, up to 17 hours, up 18 hours, up to 19hours up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours upto 24 hours or up to 1-7, 1-14, 1-21 or 1-30 days before or after thesecond therapeutic agent.

Pharmaceutical Compositions

The compounds of the invention are preferably formulated intopharmaceutical compositions for administration to a mammal, preferably,a human, in a biologically compatible form suitable for administrationin vivo. Accordingly, in an aspect, the present invention provides apharmaceutical composition comprising a compound of the invention inadmixture with a suitable diluent, carrier, or excipient.

The compounds of the invention may be used in the form of the free base,in the form of salts, solvates, and as prodrugs. All forms are withinthe scope of the invention. In accordance with the methods of theinvention, the described compounds or salts, solvates, or prodrugsthereof may be administered to a patient in a variety of forms dependingon the selected route of administration, as will be understood by thoseskilled in the art. The compounds of the invention may be administered,for example, by oral, parenteral, buccal, sublingual, nasal, rectal,patch, pump, or transdermal administration and the pharmaceuticalcompositions formulated accordingly. Parenteral administration includesintravenous, intraperitoneal, subcutaneous, intramuscular,transepithelial, nasal, intrapulmonary, intrathecal, rectal, and topicalmodes of administration. Parenteral administration may be by continuousinfusion over a selected period of time.

A compound of the invention may be orally administered, for example,with an inert diluent or with an assimilable edible carrier, or it maybe enclosed in hard- or soft-shell gelatin capsules, or it may becompressed into tablets, or it may be incorporated directly with thefood of the diet. For oral therapeutic administration, a compound of theinvention may be incorporated with an excipient and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, and wafers.

A compound of the invention may also be administered parenterally.Solutions of a compound of the invention can be prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, DMSO, and mixtures thereof with or without alcohol, and inoils. Under ordinary conditions of storage and use, these preparationsmay contain a preservative to prevent the growth of microorganisms.Conventional procedures and ingredients for the selection andpreparation of suitable formulations are described, for example, inRemington's Pharmaceutical Sciences (2003, 20th ed.) and in The UnitedStates Pharmacopeia: The National Formulary (USP 24 NF19), published in1999. The pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that may be easily administered via syringe.

A compound described herein may be administered intratumorally, forexample, as an intratumoral injection. Intratumoral injection isinjection directly into the tumor vasculature and is specificallycontemplated for discrete, solid, accessible tumors. Local, regional, orsystemic administration also may be appropriate. A compound describedherein may advantageously be contacted by administering an injection ormultiple injections to the tumor, spaced for example, at approximately,1 cm intervals. In the case of surgical intervention, the presentinvention may be used preoperatively, such as to render an inoperabletumor subject to resection. Continuous administration also may beapplied where appropriate, for example, by implanting a catheter into atumor or into tumor vasculature.

The compounds of the invention may be administered to an animal, e.g., ahuman, alone or in combination with pharmaceutically acceptablecarriers, as noted herein, the proportion of which is determined by thesolubility and chemical nature of the compound, chosen route ofadministration, and standard pharmaceutical practice.

Dosages

The dosage of the compounds of the invention, and/or compositionscomprising a compound of the invention, can vary depending on manyfactors, such as the pharmacodynamic properties of the compound; themode of administration; the age, health, and weight of the recipient;the nature and extent of the symptoms; the frequency of the treatment,and the type of concurrent treatment, if any; and the clearance rate ofthe compound in the animal to be treated. One of skill in the art candetermine the appropriate dosage based on the above factors. Thecompounds of the invention may be administered initially in a suitabledosage that may be adjusted as required, depending on the clinicalresponse. In general, satisfactory results may be obtained when thecompounds of the invention are administered to a human at a daily dosageof, for example, between 0.05 mg and 3000 mg (measured as the solidform).

Alternatively, the dosage amount can be calculated using the body weightof the patient. For example, the dose of a compound, or pharmaceuticalcomposition thereof, administered to a patient may range from 0.1-50mg/kg.

EXAMPLES

The abbreviations below are used throughout the examples section.

-   Boc tert-butoxycarbonyl-   DCM dichloromethane-   DIPEA or DIEA N.N-diisopropylethylamine-   DMF N.N-dimethylformamide-   DMSO dimethyl sulfoxide-   EDCI N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride-   EEDQ 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline-   EtOH ethyl alcohol-   h or hr hour-   HOBt or HOBT 1-hydroxybenzotriazole hydrate-   MeOH methyl alcohol-   MsCl methanesulfonyl chloride-   NaHMDS sodium bis(trimethylsilyl)amide-   PdCl₂(dtbpf)    dichloro[1,1′-bis(di-t-butylphosphino)ferrocene]palladium(II)-   THF tetrahydrofuran-   TMSCHN₂ (diazomethyl)trimethylsilane

Example 1. Preparation ofN-((S)-1-(4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-methoxy-1-oxopropan-2-yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide

N-((S)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-methoxy-1-oxopropan-2-yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamidewas synthesized as shown in Scheme 1 below.

Step 1: Preparation of 6-fluoropyridine-2-carbonyl Chloride(Intermediate B)

To a cooled (0 ° C.) solution of 6-fluoropyridine-2-carboxylic acid(50.0 g, 354 mmol) in dichloromethane (500 mL) and N,N-dimethylformamide(0.26 mL, 3.54 mmol) was added oxalyl chloride (155 mL, 1.77 mol). Aftercomplete addition of oxalyl chloride, the reaction mixture was warmed toroom temperature. After 0.5 hours, the mixture was concentrated undervacuum to give Intermediate B (56.50 g) as a white solid, which was usedin the next step without further purification.

Step 2: Preparation of 2-chloro-1-(6-fluoro-2-pyridyl)ethenone(Intermediate C)

To a cooled (0° C.) mixture of Intermediate B (56.0 g, 351 mmol) in1,4-dioxane (800 mL) was added in a dropwise manner a solution of 2Mtrimethylsilyl diazomethane in hexanes (351 mL, 702 mmol). The resultingreaction mixture was stirred at 25° C. for 10 h. The reaction mixturewas subsequently quenched with a solution of 4M HCl in 1,4-dioxane (500mL, 2.0 mol). After stirring for 2 h, the reaction solution wasconcentrated under vacuum to give an oil. The residue was diluted withsaturated aqueous NaHCO₃ and extracted three times with ethyl acetate.The combined organic layers were washed twice with brine, dried overNa2SO₄, filtered, and concentrated under reduced pressure to giveIntermediate C (35.5 g) as a white solid, which was used to next stepdirectly. LCMS (ESI) m/z: [M+H]⁺=173.8.

Step 3: Preparation of 4-(6-fluoro-2-pyridyl)thiazol-2-amine(Intermediate E)

To a solution of Intermediate C (35.5 g, 205 mmol) and thiourea (14.0 g,184 mmol) in a mixture of methanol (250 mL) and water (250 mL) at roomtemperature was added NaF (3.56 g, 84.8 mmol). After stirring for 0.5 h,the reaction mixture was partially concentrated under vacuum to removeMeOH, and the resulting solution was acidified to pH —3 with aqueous 2MHCl. After 15 minutes, the solution was extracted three times with ethylacetate. The organic layers were discarded and the aqueous phase wasalkalized with saturated aqueous NaHCO3 and stirred for 30 minutes, andextracted three times with ethyl acetate. The combined organic layerswere washed three times with brine, dried over Na₂SO₄, filtered, andconcentrated under reduced pressure. The residue was triturated withpetroleum ether and stirred at 25 ° C. for 10 minutes and filtered. Theresultant solids were dried under vacuum to give Intermediate E (28.0 g,143 mmol, 70.1% yield, 100% purity) as a white solid. LCMS (ESI) m/z:[M+H]⁺=195.8.

¹H NMR (400 MHz, DMSO-d₆) δ 8.00-7.96 (m, 1H), 7.72 (d, J=7.2 Hz, 1H),7.24 (s, 1H), 7.16 (s, 2H), 7.02 (d, J =8.0 Hz, 1H).

Step 4: Preparation of4-[6-[cis-2,6-dimethylmorpholin-4-yl]-2-pyridyl]thiazol-2-amine(Intermediate G)

Ten separate mixtures of Intermediate E (2.00 g, 10.3 mmol),cis-2,6-dimethylmorpholine (3.54 g, 30.7 mmol), and DIPEA (5.35 mL, 30.7mmol) in dimethyl sulfoxide (10 mL) were stirred in parallel at 120° C.under N2 atmosphere. After 36 h, the reaction mixtures were combined andadded dropwise to water. The resulting suspension was filtered and thefilter cake was washed three times with water and once with petroleumether, then dried over under reduced pressure to give Intermediate G(25.5 g, 87.8 mmol, 95.2% yield) as a yellow solid.

LCMS (ESI) m/z: [M+H]⁺=291.2.

¹H NMR (400 MHz, DMSO-d₆) δ 7.56-7.54 (m, 1H), 7.17 (s, 1H), 7.13 (d,J=7.6 Hz, 1H), 7.01 (s, 2H), 6.72 (d, J=8.8 Hz, 1H), 4.26-4.15 (m, 2H),3.67-3.55 (m, 2H), 2.38-2.34 (m, 2H), 1.17 (d, J=6.4 Hz, 6H).

Step 5: Preparation of tert-butylN-[(1S)-2-[[4-[6-[cis-2,6-dimethylmorpholin-4-yl]-2-pyridyl]thiazol-2-yl]amino]-1-(methoxymethyl)-2-oxo-ethyl]carbamate (Intermediate I)

To a solution of Intermediate G (12.0 g, 41.3 mmol) and(2S)-2-(tertbutoxycarbonylamino)-3-methoxy-propanoic acid (10.9 g, 49.6mmol) in dichloromethane (60 mL) was added EEDQ (12.3 g, 49.6 mmol).After stirring at room temperature for 16 h, the reaction mixture wasconcentrated under reduced pressure to give a residue. The residue waspurified by silica gel column chromatography (petroleum ether:ethylacetate=2:1 to 3:2) to give Intermediate 1 (20.0 g, 40.7 mmol, 98.5%yield) as a yellow gum.

LCMS (ESI) m/z: [M+H]+=492.2.

¹H NMR (400 MHz, DMSO-d₆) δ 12.37 (s, 1H), 7.78 (s, 1H), 7.64-7.60 (m,1H), 7.25 (d, J=7.2 Hz, 1H), 7.16 (d, J=7.2 Hz, 1H), 6.79 (d, J 32 8.4Hz, 1H), 4.50-4.48 (m, 1H), 4.25 (d, J=11.6 Hz, 2H), 3.70-3.51 (m, 4H),3.26 (s, 3H), 2.44-2.40 (m, 2H), 1.39 (s, 9H), 1.18 (d, J=6.4 Hz, 6H).

Step 6: Preparation of(S)-4-(4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2yl7-1-methoxy-7-3-oxobutan-2-aminium chloride (Intermediate J)

To a solution of 4M HCl in 1,4-dioxane (200 mL, 800 mmol) was added asolution of Intermediate I (20.0 g, 40.7 mmol) in dichloromethane (50mL). After stirring at room temperature for 2 h, the mixture was dilutedwith methyl tert-butyl ether resulting in a suspension. The solid wascollected by filtration, washed twice with methyl tert-butyl ether, anddried in vacuo to give Intermediate J (19.0 g) as a yellow solid, whichwas used in the next step without further purification.

LCMS (ESI) m/z: [M+H]⁺=392.3.

¹H NMR (400 MHz, DMSO-d₆) δ 13.44-12.30 (m, 1H), 8.65 (d, J=4.4 Hz, 3H),7.87 (s, 1H), 7.66-7.64 (m, 1H), 7.25 (d, J 32 7.2 Hz, 1H), 6.83 (d,J=8.8 Hz, 1H), 4.39-4.30 (m, 1H), 4.25 (d, J=11.6 Hz, 2H), 3.94-3.86 (m,1H), 3.85-3.77 (m, 1H), 3.69-3.57 (m, 2H), 3.31 (s, 3H), 2.43 (m, 2H),1.18 (d, J=6.4 Hz, 6H).

Preparation of 1-(methylsulfonyl)-1H-pyrrole-3-carboxylic acid(Intermediate K)

1-(methylsulfonyI)-1H-pyrrole-3-carboxylic acid was synthesized as shownin Scheme 2 below.

Step A: Preparation of tert-butyl 1H-pyrrole-3-carboxylate (IntermediateN)

To a mixture of tert-butyl-prop-2-enoate (78.6 mL, 542 mmol) and1-(isocyanomethylsulfonyl)-4-methylbenzene (106 g, 542 mmol) in THF(1300 mL) was added 60% NaH in mineral oil (25.97 g, 649 mmol) slowly at30° C. over 1 hour and then heated to 70° C. After 2 h, the reactionmixture was poured into saturated aqueous NH₄Cl solution and extractedthree times with ethyl acetate. The combined organic phase was washedtwice with brine, dried over anhydrous Na₂SO₄, filtered, andconcentrated under reduced pressure to afford a residue. The residue waspurified by silica gel column chromatography (petroleum ether:ethylacetate=20:1 to 3:1) to afford Intermediate N (41.5 g, 236 mmol, 43%yield) as a yellow solid.

LCMS (ESI) m/z [M+Na]⁺=180.4.

¹H NMR (400 MHz, CDCl₃) δ 8.36 (br s, 1H), 7.35-7.25 (m, 1H), 6.71 -6.62 (m, 1 H), 6.59-6.49 (m, 1H), 1.48 (s, 9H).

Step B: Preparation of tert-butyl 1-methylsulfonylpyrrole-3-carboxylate(Intermediate O)

To a cooled solution (0° C.) of Intermediate N (40.5 g, 242 mmol) in THF(1500 mL) was added a 1M solution of NaHMDS (484 mL, 484 mmol). Afterstirring at 0° C. for 30 min, methanesulfonyl chloride (28.1 mL, 363mmol) was slowly added and the mixture was warmed to 30° C. After 16 h,the reaction mixture was slowly poured into saturated aqueous NH₄Clsolution and extracted three times with ethyl acetate. The combinedorganic layers were washed twice with brine, dried over anhydrousNa₂SO₄, filtered, and concentrated under reduced pressure to afford aresidue. The residue was purified by silica gel chromatography(petroleum ether:ethyl acetate=10:1) to afford a yellow solid. Theyellow solid was triturated with methyl tert-butyl ether at roomtemperature, stirred for 20 minutes, filtered, and dried in vacuum toafford Intermediate O (25.7 g, 105 mmol, 43% yield) as a white solid.

¹H NMR (400 MHz, CDCl₃) δ 7.66-7.64 (m, 1H), 7.10-7.08 (m, 1H),6.73-6.71 (m, 1H), 3.21 (s, 3H), 1.56 (s, 9H).

Step C: Preparation of 1-methylsulfonylpyrrole-3-carboxylic acid(Intermediate K)

To a mixture of Intermediate O (25.7 g, 105 mmol) in 1,4-dioxane (100mL) was added a 4M solution of HCl in 1,4-dioxane (400 mL, 1.6 mol) at15° C. After stirring at at 15° C. for 14 h, the reaction mixture wasconcentrated under reduced pressure to afford a residue. The residue wastriturated with methyl tert-butyl ether at 15° C. for 16 h. The mixturewas filtered and dried in vacuum to afford

Intermediate K (18.7 g, 98.8 mmol, 94% yield) as a white solid.

LCMS (ESI) m/z [M+H]⁺=189.8.

¹H NMR (400 MHz, methanol-d₄) δ 7.78-7.77 (m, 1H), 7.25-7.23 (m, 1H),6.72-6.70 (m, 1H), 3.37 (s, 3H).

Step 7: Preparation ofN-((S)-1((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-methoxy-1-oxopropan-2-yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide

To a solution of 1-methylsulfonylpyrrole-3-carboxylic acid (IntermediateK) (2.43 g, 12.9 mmol), EDCI (2.69 g, 14.0 mmol), HOBt (1.89 g, 14.0mmol), and DIPEA (10.2 mL, 58.4 mmol) in dichloromethane (50 mL) wasadded Intermediate J (5.00 g, 11.7 mmol). After stirring at roomtemperature for 4 h, the reaction mixture was concentrated under reducedpressure. The residue was diluted with water and extracted three timeswith ethyl acetate. The combined organic layers were washed three timeswith saturated aqueous NH₄Cl, once with brine, dried over Na₂SO₄,filtered, and concentrated under reduced pressure to give a residue. Theresidue was purified by silica gel column chromatography (petroleumether:ethyl acetate=1:1 to 1:2). The residue was triturated with methyltert-butyl ether. After 0.5 h, the suspension was filtered, the filtercake was washed with methyl tert-butyl ether, and dried in vacuo. Thesolid was dissolved in dimethyl sulfoxide (12 mL) and added dropwise towater (800 mL). The suspension was filtered to give wet filter cake. Thefilter cake was suspended in water and stirred at room temperature.After 1 hour, the solid was collected by filtration, washed three timeswith water and dried in vacuo to giveN-((S)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-methoxy-1-oxopropan-2-yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide(3.9 g, 6.93 mmol, 59.3% yield) as a white solid.

LCMS (ESI) m/z: [M+H]⁺=563.1.

¹H NMR (400 MHz, DMSO-d₆) δ 12.49 (br s, 1H), 8.51 (d, J =7.2 Hz, 1H),7.98-7.97 (m, 1H), 7.78 (s, 1H), 7.67-7.57 (m, 1H), 7.29-7.27 (m, 1H),7.26 (d, J=7.2 Hz, 1H), 6.88-6.74 (m, 2H), 4.94-4.91 (m, 1H), 4.25 (d,J=11.6 Hz, 2H), 3.77-3.67 (m, 2H), 3.63-3.62 (m, 2H), 3.57 (s, 3H), 3.31(s, 3H), 2.44-2.38 (m, 2H), 1.18 (d, J=6.0 Hz, 6H).

Example 2. Preparation ofN-((S)-14(4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yOthiazol-2-yl)amino)-3-(methoxy-d₃)-1-oxopropan-2-yl-3,3-d₂)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide

N-((S)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-(methoxy-d₃)-1-oxopropan-2-yl-3,3-d₂)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamidewas prepared according to the synthetic protocol described in Example 1with Intermediate H replaced withN-(tert-Butoxycarbonyl)-O-(methyl-d₃)-L-serine-3,3-d₂.N-(tert-Butoxycarbonyl)-O-(methyl-d₃)-L-serine-3,3-d₂ was prepared fromisotopically enriched material according to synthetic proceduresdescribed in A. Yang et al, Org. Process Res. Dev. 2019, 23, 818-824.

LCMS (ESI) m/z: [M+H]⁺=568.2.

¹H NMR (400 MHz, DMSO-d₆) δ 12.45 (s, 1H), 8.47 (d, J=7.2 Hz, 1H), 7.98(dd, J=2.3, 1.7 Hz, 1H), 7.78 (s, 1H), 7.62 (dd, J=8.5, 7.4 Hz, 1H),7.29 (dd, J=3.2, 2.3 Hz, 1H), 7.26 (d, J=7.3 Hz, 1H), 6.84-6.75 (m, 2H),4.91 (d, J=7.2 Hz, 1H), 4.25 (dd, J=13.1, 2.3 Hz, 2H), 3.69-3.59 (m,2H), 3.56 (s, 3H), 2.42 (dd, J=12.8, 10.5 Hz, 2H), 1.18 (d, J=6.2 Hz,6H).

Example 3. Preparation ofN-((R)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yy)thiazol-2-yl)amino)-3-(methoxy)-1-oxopropan-2-yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide

N-((R)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-(methoxy)-1-oxopropan-2-yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamidewas prepared according to the synthetic protocol described in Example 1with Intermediate H replaced with(2R)-2-(tertbutoxycarbonylamino)-3-methoxy-propanoic acid.

LCMS (ESI) m/z: [M+H]⁺=563.1.

¹H NMR (400 MHz, DMSO-d₆) δ 12.5 (s, 1H), 8.50 (d, J=7.2 Hz, 1H), 7.98(t, J=1.6 Hz, 1H), 7.78 (s, 1H), 7.62 (dd, J=7.2, 8.4 Hz, 1H), 7.29 (dd,J=2.0, 3.2 Hz, 1H), 7.26 (d, J=7.2 Hz, 1H), 6.79-6.81 (m, 2H), 4.92 (q,J=6.4, 12.8 Hz, 1H), 4.25 (d, J=11.2 Hz, 2H), 3.69-3.75 (m, 2H),3.59-3.66 (m, 2H), 3.56 (s, 3H), 3.31 (s, 3H), 2.41 (dd, J=10.8, 12.8Hz, 2H), 1.18 (d, J=6.0 Hz, 6H).

Example 4. Preparation ofN-((R)-14(4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-(methoxy-d₃)-1-oxopropan-2-yl-3,3-d₂)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide

N-((R)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-(methoxy-d₃)-1-oxopropan-2-yl-3,3-d₂)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamidewas prepared according to the synthetic protocol described in Example 1with Intermediate H replaced withN-(tert-Butoxycarbonyl)—O—(methyl-d₃)-D-serine-3,3-d₂.N-(tert-Butoxycarbonyl)—O—(methyl-d₃)-D-serine-3,3-d₂ was prepared fromisotopically enriched material according to synthetic proceduresdescribed in A. Yang et al, Org. Process Res. Dev. 2019, 23, 818-824.

LCMS (ESI) m/z: [M+H]⁺=568.3.

¹H NMR (400 MHz, DMSO-d₆) δ 12.46 (s, 1H), 8.52- 8.38 (m, 1H), 7.97 (t,J=1.9 Hz, 1H), 7.76 (s, 1H), 7.62 (dd, J=8.5, 7.3 Hz, 1H), 7.29 (dd,J=3.3, 2.3 Hz, 1H), 7.26 (d, J=7.4 Hz, 1H), 6.79 (dt, J=5.1, 1.8 Hz,2H), 4.89 (d, J=5.2 Hz, 1H), 4.31-4.20 (m, 2H), 3.63 (ddd, J=10.5, 6.2,2.5 Hz, 2H), 3.56 (s, 3H), 2.41 (dd, J=12.8, 10.5 Hz, 2H), 1.18 (d,J=6.2 Hz, 6H).

Example 5. Assay for ATPase Catalytic Activity of BRM and BRG-1

The ATPase catalytic activity of BRM or BRG-1 was measured by an invitro biochemical assay using ADP-Glo™ (Promega, V9102). The ADP-Glo™kinase assay was performed in two steps once the reaction was complete.The first step is to deplete any unconsumed ATP in the reaction. Thesecond step was to convert the reaction product ADP to ATP, which willbe utilized by the luciferase to generate luminesce and be detected by aluminescence reader, such as Envision.

The assay reaction mixture (10 μL) contained 30 nM of BRM or BRG-1, 20nM salmon sperm DNA (from Invitrogen, UltraPure™ Salmon Sperm DNASolution, cat #15632011), and 400 μM of ATP in the ATPase assay buffer,which comprises of 20 mM Tris, pH 8, 20 mM MgCl₂, 50 mM NaCl, 0.1%Tween-20, and 1 mM fresh DTT (Pierce™ DTT (Dithiothreitol), cat #20290).The reaction was initiated by the addition of the 2.5 μL ATPase solutionto 2.5 μL ATP/DNA solution on low volume white Proxiplate-384 plus plate(PerkinElmer, cat #6008280) and incubated at room temperature for 1hour. Then following addition of 5 μL of ADP-Glo™ Reagent provided inthe kit, the reaction incubated at room temperature for 40 minutes. Then10 μL of Kinase Detection Reagent provided in the kit was added toconvert ADP to ATP, and the reaction incubated at room temperature for60 minutes. Finally, luminescence measurement is collected with aplate-reading luminometer, such as Envision.

BRM and BRG-1 were synthesized from high five insect cell lines with apurity of greater than 90%.

N-((S)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-methoxy-1-oxopropan-2-yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamidewas found to have an IP₅₀ of 3.9 nM against BRM and 5.2 nM against BRG1in the assay.N-((R)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-(methoxy-d₃)-1-oxopropan-2-yl-3,3-d₂)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamidewas found to have an IP₅₀ of 443 nM against BRM and 777 nM against BRG1in the assay.N-((S)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-(methoxy-d₃)-1-oxopropan-2-yl-3,3-d₂)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide was found to have an IP₅₀ of 4.6 nM against BRMand 7.4 nM against BRG1 in the assay.

Example 6. Synthesis of Compound A

BRG1/BRM Inhibitor Compound A has the structure:

Compound A was synthesized as shown in Scheme 3 below.

The ATPase catalytic activity of BRM or BRG-1 in the presence ofCompound A was measured by the in vitro biochemical assay using ADP-Glo™(Promega, V9102) described above. Compound A was found to have an IP₅₀of 10.4 nM against BRM and 19.3 nM against BRG1 in the assay.

Example 7. Effects of BRG1/BRM ATPase Inhibition on the Growth of UvealMelanoma and Hematological Cancer Cell Lines

Procedure: Uveal melanoma cell lines (92-1, MP41, MP38, MP46), prostatecancer cell lines (LNCAP), lung cancer cell lines (NCI-H1299), andimmortalized embryonic kidney lines (HEK293T) were plated into 96 wellplates with growth media (see Table 1). BRG1/BRM ATPase inhibitor,Compound A, was dissolved in DMSO and added to the cells in aconcentration gradient from 0 to 10 μM at the time of plating. Cellswere incubated at 37° C. for 3 days. After three days of treatment, themedia was removed from the cells, and 30 microliters of TrypLE (Gibco)was added to cells for 10 minutes. Cells were detached from the plates,and resuspended with the addition of 170 microliters of growth media.Cells from two DMSO-treated control wells were counted, and the initialnumber of cells plated at the start of the experiment, were re-platedinto fresh-compound containing plates for an additional four days at 37°C. At day 7, cells were harvested as described above. On day 3 and day7, relative cell growth was measured by the addition of Cell-titer glo(Promega), and luminescence was measured on an Envision plate reader(Perkin Elmer). The concentration of compound at which each cell line'sgrowth was inhibited by 50% (GI₅₀), was calculated using Graphpad Prism,and is plotted below. For multiple myeloma cell lines (OPM2, MM1S, LP1),ALL cell lines (TALL1, JURKAT, RS411), DLBCL cell lines (SUDHL6, SUDHL4,DB, WSUDLCL2, PFEIFFER), AML cell lines (OCIAML5), MDS cell lines(SKM1), ovarian cancer cell lines (OV7, TYKNU), esophageal cancer celllines (KYSE150), rhabdoid tumor lines (RD, G402, G401, HS729, A204),liver cancer cell lines (HLF, HLE, PLCRPF5), and lung cancer cell lines(SW1573, NCIH2444), the above methods were performed with the followingmodifications: Cells were plated in 96 well plates, and the next day,BRG1/BRM ATPase inhibitor, Compound A, was dissolved in DMSO and addedto the cells in a concentration gradient from 0 to 10 μM. At the time ofcell splitting on days 3 and 7, cells were split into new 96 wellplates, and fresh compound was added four hours after re-plating. Table1 lists the tested cell lines and growth media used.

TABLE 1 Cell Lines and Growth Media Cell Line Source Growth Media 92-1SIGMA RPMI1640 + 20% FBS A204 ATCC McCoy's 5A + 10% FBS DB ATCCRPMI1640 + 10% FBS G401 ATCC McCoy's 5A + 10% FBS G402 ATCC McCoy's 5A +10% FBS HEK293T ATCC DMEM + 10% FBS HLE JCRB DMEM + 10% FBS HLF JCRBDMEM + 10% FBS H5729 ATCC DMEM + 10% FBS JURKAT ATCC RPMI1640 + 10% FBSKYSE150 DSMZ RPMI1640/Ham's F12 + 10% FBS LNCAP ATCC RPMI1640 + 10% FBSLP1 DSMZ IMDM + 20% FBS MM1S ATCC RPMI1640 + 10% FBS MP38 ATCCRPMI1640 + 20% FBS MP41 ATCC RPMI1640 + 20% FBS MP46 ATCC RPMI1640 + 20%FBS NCIH1299 ATCC RPMI1640 + 10% FBS NCIH2444 ATCC RPMI1640 + 20% FBSOCIAML5 DSMZ alpha-MEM + 20% FBS + 10 ng/ml GM-CSF OPM2 DSMZ RPMI1640 +10% FBS OV7 ECACC DMEM/Ham's F12 (1:1) + 2 mM Glutamine + 10% FBS + 0.5ug/ml hydrocortisone + 10 ug/ml insulin PFEIFFER ATCC RPMI1640 + 10% FBSPLCPRF5 ATCC EMEM + 10% FBS RD ATCC DMEM + 10% FBS RS411 ATCC RPMI1640 +10% FBS SKM1 JCRB RPMI1640 + 10% FBS SUDHL4 DSMZ RPMI1640 + 10% FBSSUDHL6 ATCC RPMI1640 + 20% FBS SW1573 ATCC DMEM + 10% FBS TALL1 JCRBRPMI1640 + 10% FBS TYKNU JCRB EMEM + 20% FBS WSUDLCL2 DSMZ RPMI1640 +10% FBS

Results: As shown in FIG. 1 , the uveal melanoma and hematologic cancercell lines were more sensitive to BRG1/BRM inhibition than the othertested cell lines. Inhibition of the uveal melanoma and hematologiccancer cell lines was maintained through day 7.

Example 8. Comparison of BRG1/BRM Inhibitors to Clinical PKC and MEKInhibitors in Uveal Melanoma Cell Lines

Procedure: Uveal melanoma cell lines, 92-1 or MP41, were plated in 96well plates in the presence of growth media (see Table 1). BAF ATPaseinhibitors (Compound A), PKC inhibitor (LXS196; MedChemExpress), or MEKinhibitor (Selumetinib; Selleck Chemicals) were dissolved in DMSO andadded to the cells in a concentration gradient from 0 to 10 μM at thetime of plating. Cells were incubated at 37° C. for 3 days. After threedays of treatment, cell growth was measured with Cell-titer glow(Promega), and luminescence was read on an Envision plate reader (PerkinElmer). Results: As shown in FIG. 2 and FIG. 3 , Compound A showedcomparable growth inhibition of uveal melanoma cells as the clinical PKCand MEK inhibitors. Further, Compound A was found to result in a fasteronset of inhibition than the clinical PKC and MEK inhibitors.

Example 9. Synthesis of Compound B

BRG1/BRM Inhibitor Compound B has the structure:

Compound B was synthesized as shown in Scheme 4 below.

To a mixture of(2S)-2-amino-4-methylsulfanyl-N-[4-[3-(4-pyridyl)phenyl]thiazol-2-yl]butanamide(2 g, 4.75 mmol, HCl salt) and 1-methylsulfonylpyrrole-3-carboxylic acid(898.81 mg, 4.75 mmol) in DMF (20 mL) was added EDCI (1.37 g, 7.13mmol), HOBt (962.92 mg, 7.13 mmol), and DIEA (2.46 g, 19.00 mmol, 3.31mL) and the mixture was stirred at 25° C. for 3 h. The mixture waspoured into H₂O (100 mL) and the precipitate was collected byfiltration. The solid was triturated in MeOH (20 mL) and the precipitatewas collected by filtration. The solid was dissolved in DMSO (10 mL) andthen the mixture was poured into MeOH (50 mL), and the formedprecipitate was collected by filtration and lyophilized to give CompoundB (2.05 g, 3.66 mmol, 77.01% yield) as a white solid.

LCMS (ESI) m/z [M+H]⁺=555.9.

¹H NMR (400 MHz, DMSO) δ 12.49 (s, 1H), 8.68-8.66 (m, 2H), 8.46 (d,J=7.2 Hz, 1H), 8.31-8.30 (m, 1H), 8.02-8.00 (m, 1H), 7.94-7.96 (m, 1H),7.83 (s, 1H), 7.73-7.74 (m, 3H), 7.61-7.57 (m, 1H), 7.31-7.29 (m, 1H),6.79-6.77 (m, 1H), 4.74-4.69 (m, 1H), 3.57 (s, 3H), 2.67-2.53 (m, 2H),2.13-2.01 (m, 5H). ee %=100%.

Compound B was found to have an IP₅₀ of 3.6 nM against BRM and 5.7 nMagainst BRG1 in the ATPase assay described.

Example 10. Effects of BRG1/BRM ATPase Inhibition on the Growth of UvealMelanoma, Hematological Cancer, Prostate Cancer, Breast Cancer, andEwing's Sarcoma Cell Lines

Procedure: All cell lines described above in Example 7 were also testedas described above with Compound B. In addition, the following celllines were also tested as follows. Briefly, for Ewing's sarcoma celllines (CADOES1, RDES, SKES1), retinoblastoma cell lines (WERIRB1), ALLcell lines (REH), AML cell lines (KASUMI1), prostate cancer cell lines(PC3, DU145, 22RV1), melanoma cell lines (SH4, SKMEL28, WM115, COL0829,SKMEL3, A375), breast cancer cell lines (MDAMB415, CAMA1, MCF7, BT474,HCC1419, DU4475, BT549), B-ALL cell lines (SUPB15), CML cell lines(K562, MEG01), Burkitt's lymphoma cell lines (RAMOS2G64C10, DAUDI),mantle cell lymphoma cell lines (JEKO1, REC1), bladder cancer cell lines(HT1197), and lung cancer cell lines (SBC5), the above methods wereperformed with the following modifications: Cells were plated in 96 wellplates, and the next day, BRG1/BRM ATPase inhibitor, Compound B, wasdissolved in DMSO and added to the cells in a concentration gradientfrom 0 to 10 μM. At the time of cell splitting on days 3 and 7, cellswere split into new 96 well plates, and fresh compound was added fourhours after re-plating. Table 2 lists the tested cell lines and growthmedia used.

TABLE 2 Cell Lines And Growth Media Cell Line Source Growth Media 22RV1ATCC RPMI1640 + 10% FBS A375 ATCC DMEM + 10% FBS BT474 ATCC Hybricaremedium + 1.5 g/L sodium bicarbonate + 10% FBS BT549 ATCC RPMI1640 +0.023 IU/ml insulin + 10% FBS CADOES1 DSMZ RPMI1640 + 10% FBS CAMA1 ATCCEMEM + 10% FBS COLO829 ATCC RPMI1640 + 10% FBS DAUDI ATCC RPMI1640 + 10%FBS DU145 ATCC EMEM + 10% FBS DU4475 ATCC RPMI1640 + 10% FBS HCC1419ATCC RPMI1640 + 10% FBS HT1197 ATCC EMEM + 10% FBS JEKO1 ATCC RPMI1640 +20% FBS K562 ATCC IMDM + 10% FBS KASUMI1 ATCC RPMI1640 + 10% FBS MCF7ATCC EMEM + 0.01 mg/ml bovine insulin + 10% FBS MDAM B415 ATCCLeibovitz's L-15 + 2 mM L-glutamine + 10 mcg/ml insulin + 10 mcg/mlglutathione + 15% FBS MEG01 ATCC RPMI1640 + 10% FBS PC3 ATCC F-12K + 10%FBS RAMOS2G64C10 ATCC RPMI1640 + 10% FBS RDES ATCC RPMI1640 + 15% FBSREC1 ATCC RPMI1640 + 10% FBS REH ATCC RPMI1640 + 10% FBS SBC5 JCRBEMEM + 10% FBS SH4 ATCC DMEM + 10% FBS SKES1 ATCC McCoy's 5A + 15% FBSSKMEL28 ATCC EMEM + 10% FBS SKMEL3 ATCC McCoy's 5A + 15% FBS SUPB15 ATCCIMDM + 4 mM L-glutamine + 1.5 g/L sodium bicarbonate + 0.05 mM2-mercaptoethanol + 20% FBS WERIRB1 ATCC RPMI1640 + 10% FBS WM115 ATCCEMEM + 10% FBS

Results: As shown in FIG. 4 , the uveal melanoma, hematologic cancer,prostate cancer, breast cancer, and Ewing's sarcoma cell lines were moresensitive to BRG1/BRM inhibition than the other tested cell lines.Inhibition of the uveal melanoma, hematologic cancer, prostate cancer,breast cancer, and

Ewing's sarcoma cell lines was maintained through day 7.

Example 11. Effects of BRG1/BRM ATPase Inhibition on the Growth ofCancer Cell Lines

Procedure: A pooled cell viability assay was performed using PRISM(Profiling Relative Inhibition Simultaneously in Mixtures) as previouslydescribed (“High-throughput identification of genotype-specific cancervulnerabilities in mixtures of barcoded tumor cell lines”, Yu et al,Nature Biotechnology 34, 419-423, 2016), with the followingmodifications. Cell lines were obtained from the Cancer Cell Line

Encyclopedia (CCLE) collection and adapted to RPMI-1640 medium withoutphenol red, supplemented with 10% heat-inactivated fetal bovine serum(FBS), in order to apply a unique infection and pooling protocol to sucha big compendium of cell lines. A lentiviral spin-infection protocol wasexecuted to introduce a 24 nucleotide-barcode in each cell line, with anestimated multiplicity of infection (MOI) of 1 for all cell lines, usingblasticidin as selection marker. Over 750 PRISM cancer cell lines stablybarcoded were then pooled together according to doubling time in poolsof 25. For the screen execution, instead of plating a pool of 25 celllines in each well as previously described (Yu et al.), all the adherentor all the suspension cell line pools were plated together using T25flasks (100,000 cells/flask) or 6-well plates (50,000 cells/well),respectively. Cells were treated with either DMSO or compound in a8-point 3-fold dose response in triplicate, starting from a topconcentration of 10 μM. As control for assay robustness, cells weretreated in parallel with two previously validated compounds, the pan-Rafinhibitor AZ-628, and the proteasome inhibitor bortezomib, using a topconcentration of 2.5 μM and 0.039 μM, respectively.

Following 3 days of treatment with compounds, cells were lysed, genomicDNA was extracted, barcodes were amplified by PCR and detected withNext-Generation Sequencing. Cell viability was determined by comparingthe counts of cell-line specific barcodes in treated samples to those inthe

DMSO-control and Day 0 control. Dose-response curves were fit for eachcell line and corresponding area under the curves (AUCs) were calculatedand compared to the median AUC of all cell lines (FIG. 5 ).

Results: Cell lines with AUCs less than the median were considered mostsensitive.

Example 12. Effects of BRG1/BRM ATPase Inhibitors on the Growth of UvealMelanoma Cell Lines

Procedure: Uveal melanoma cell lines (92-1, MP41, MP38, MP46) andnon-small cell lung cancer cells (NCIH1299) were plated into 96 wellplates with growth media (see Table 2). BRG1/BRM ATPase inhibitor,Compound B, was dissolved in DMSO and added to the cells in aconcentration gradient from 0 to 10 μM at the time of plating. Cellswere incubated at 37° C. for 3 days. After three days of treatment, cellgrowth was measured with Cell-titer glow (Promega), and luminescence wasread on an Envision plate reader (Perkin Elmer).

Results: As shown in FIG. 6 , Compound B resulted in potent growthinhibition in the cell lines.

Example 13. Comparison of BRG1/BRM Inhibitors to Clinical PKC and MEKInhibitors in Uveal Melanoma Cell Lines

Procedure: Uveal melanoma cell lines, 92-1 or MP41, were plated in 96well plates in the presence of growth media (see Table 2). BAF ATPaseinhibitor (Compound B), PKC inhibitor (LXS196; MedChemExpress), and MEKinhibitor (Selumetinib; Selleck Chemicals) were dissolved in DMSO andadded to the cells in a concentration gradient from 0 to 10 μM at thetime of plating. Cells were incubated at 37° C. for 3 days. After threedays of treatment, cell growth was measured with Cell-titer glow(Promega), and luminescence was read on an Envision plate reader (PerkinElmer).

Results: As shown in FIG. 7 and FIG. 8 , Compound B showed more potenteffects on growth inhibition of uveal melanoma cells as compared to theclinical PKC and MEK inhibitors. Further, Compound B was found to resultin a faster onset of growth inhibition than the clinical PKC and MEKinhibitors.

Example 14. BRG1/BRM ATPase Inhibitors are effective at Inhibiting theGrowth of PKC Inhibitor-resistant Cells

Procedure: MP41 uveal melanoma cells were made resistant to the PKCinhibitor (LXS196; MedChemExpress), by long-term culture in growth media(see Table 2) containing increasing concentrations of the compound, upto 1 μM. After 3 months, sensitivity of the parental MP41 cells and thePKC inhibitor (PKCi)-resistant cells to the PKC inhibitor (LXS196) orthe BRG1/BRM ATPase inhibitor (Compound B) was tested in a 7-day growthinhibition assay as described above in Example 6.

Results: While the PKCi-resistant cells could tolerate growth at higherconcentrations of LXS196 than could the parental MP41 cell line (FIG. 9), the BRG1/BRM ATPase inhibitor (Compound B) resulted in strong growthinhibition of both the PKCi-resistant and parental cell lines (FIG. 10). The PKCi-resistant cells were more sensitive to Compound B than werethe parental MP41 cells (FIG. 10 ).

Example 15. Synthesis of Compound C

BRG1/BRM Inhibitor Compound C has the structure:

Compound C was synthesized as shown in Scheme 5 below.

Compound C was found to have an IP₅₀ of 5.3 nM against BRM and 1.3 nMagainst BRG1 in the ATPase assay described above.

Example 16. BRG1/BRM ATPase Inhibitors Cause Uveal Melanoma Tumor GrowthInhibition In Vivo

Procedure: Nude mice (Envigo) were engrafted subcutaneously in theaxillary region with 5×10⁶ 92-1 uveal melanoma cells in 50% Matrigel.Tumors were grown to a mean of ˜200 mm³, at which point mice weregrouped and dosing was initiated. Mice were dosed once daily by oralgavage with vehicle (20% 2-Hydroxypropyl-β-Cyclodextrin) or increasingdoses of Compound C. Tumor volumes and body weights were measured overthe course of 3 weeks, and doses were adjusted by body weight to achievethe proper dose in terms of mg/kg. At this time, animals weresacrificed, and tumors were dissected and imaged.

Results: As shown in FIG. 11 and FIG. 12 , treatment with Compound C ledto tumor growth inhibition in a dose-dependent manner with tumorregression observed at the highest (50 mg/kg) dose. As shown in FIG. 13, all treatments were well tolerated with no body weight loss observed(FIG. 13 ).

Example 17. Effects of BRG1/BRM ATPase Inhibition on the Growth of UvealMelanoma and Hematological Cancer Cell Lines

Procedure: Uveal melanoma cell lines (92-1, MEL202, MP41, MP38, MP46),prostate cancer cells (22RV1), acute leukemia cells (EOL1, THP1), andhistocytic lymphoma cells (U937) were plated into 96 well plates withgrowth media (see Table 2). BRG1/BRM ATPase inhibitor,N-((S)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-methoxy-1-oxopropan-2-yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide,was dissolved in DMSO and added to the cells in a concentration gradientfrom 0 to 2 μM (for uveal melanoma cell lines), or 0 to 1 μM (for othercell lines), at the time of plating. Cells were incubated at 37° C. for3 days. After three days of treatment, cell growth was measured withCell-titer glow (Promega), and luminescence was read on an Envisionplate reader (Perkin Elmer).

Results: As shown in FIG. 14 ,N-((S)-1-(4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-methoxy-1-oxopropan-2-yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamideresulted in potent growth inhibition in all the cell lines. As shown inTable 3, measured absolute ICso values were below 350 nanomolar for allcell lines tested.

Table 3 lists the tested cell lines, growth media used, and absoluteIC₅₀ values (nM) after 3 days of compound treatment.

TABLE 3 Cell Lines, Growth Media, and Absolute IC₅₀ values Cell AbsoluteLine Source Growth Media Cancer Type IC₅₀ (nM) 22RV1 ATCC RPMI1640 + 10%Prostate 29.7 FBS 92-1 SIGMA RPMI1640 + 10% Uveal melanoma 0.3 FBS EOL1DSMZ RPMI1640 + 10% Acute myeloid 75.5 FBS leukemia MEL202 SIGMARPMI1640 + 10% Uveal melanoma 62.3 FBS MP38 ATCC RPMI1640 + 20% Uvealmelanoma 31.5 FBS MP41 ATCC RPMI1640 + 20% Uveal melanoma 11.8 FBS MP46ATCC RPMI1640 + 20% Uveal melanoma 112.6 FBS THP1 ATCC RPMI1640 + 10%Acute monocytic 344.9 FBS leukemia U937 ATCC RPMI1640 + 10% Histiocytic14.8 FBS lymphoma

Example 18. BRG1/BRM ATPase Inhibition causes Uveal Melanoma TumorGrowth Inhibition In Vivo

Procedure: Nude mice (Envigo) were engrafted subcutaneously in theaxillary region with 5×10⁶ 92-1 uveal melanoma cells in 50% Matrigel.Tumors were grown to a mean of ˜200 mm³, at which point mice weregrouped and dosing was initiated. Mice were dosed once daily by oralgavage with vehicle (20% 2-Hydroxypropyl-β-Cyclodextrin) or increasingdoses of N-((S)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-methoxy-1-oxopropan-2-yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide.Tumor volumes and body weights were measured over the course of 3 weeks,and doses were adjusted by body weight to achieve the proper dose interms of mg/kg.

Results: As shown in FIG. 15 , treatment withN-((S)-1(4(4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-methoxy-1-oxopropan-2-yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamideled to tumor growth inhibition in a dose-dependent manner with tumorregression observed at the highest (1.5 mg/kg) dose. As shown in FIG. 16, all treatments were well tolerated based on % body weight changeobserved.

Other Embodiments

While the invention has been described in connection with specificembodiments thereof, it will be understood that invention is capable offurther modifications and this application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth, and follows in the scope ofthe claims.

Other embodiments are in the claims.

What is claimed is:
 1. A method of treating metastatic uveal melanoma ina subject in need thereof, the method comprising administering to thesubject an effective amount of a compound having the formula (A):

or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the compound of formula (A) has the structure:

or a pharmaceutically acceptable salt thereof.
 3. The method of claim 2,wherein the compound of formula (A) has the structure:

or a pharmaceutically acceptable salt thereof.
 4. The method of claim 1,wherein the compound of formula (A) has the structure:

or a pharmaceutically acceptable salt thereof.
 5. The method of claim 1,wherein the method comprises administering a pharmaceutical compositioncomprising the compound of formula (A) having the structure:

or a pharmaceutically acceptable salt thereof, wherein each substituentD is a position that is deuterium enriched within the pharmaceuticalcomposition.
 6. The method of claim 5, wherein the pharmaceuticalcomposition comprises the compound of formula (A) having the structure:

or a pharmaceutically acceptable salt thereof, wherein each substituentD is a position that is deuterium enriched within the pharmaceuticalcomposition.
 7. The method of claim 6, wherein the pharmaceuticalcomposition comprises the compound of formula (A) having the structure:

or a pharmaceutically acceptable salt thereof, wherein each substituentD is a position that is deuterium enriched within the pharmaceuticalcomposition.
 8. The method of claim 5, wherein the pharmaceuticalcomposition comprises the compound of formula (A) having the structure:

or a pharmaceutically acceptable salt thereof, wherein each substituentD is a position that is deuterium enriched within the pharmaceuticalcomposition.
 9. The method of claim 1, wherein the metastatic uvealmelanoma expresses BRG1 and/or BRM protein.
 10. The method of claim 1,wherein the subject or metastatic uveal melanoma has an BRG1 loss offunction mutation.
 11. The method of claim 10, wherein the BRG1 loss offunction mutation is in the ATPase catalytic domain of the protein. 12.The method of claim 10, wherein the BRG1 loss of function mutation is adeletion at the C-terminus of BRG1.
 13. The method of claim 1, whereinthe metastatic uveal melanoma does not have, or has been determined notto have, an epidermal growth factor receptor mutation and/or ananaplastic lymphoma kinase driver mutation.
 14. The method claim 1,wherein the metastatic uveal melanoma has, or has been determined tohave, a KRAS mutation, a mutation in GNAQ, a mutation in GNA11, amutation in PLCB4, a mutation in CYSLTR2, a mutation in BAP1, a mutationin SF3B1, a mutation in EIF1AX, a TFE3 translocation, a TFEBtranslocation, a MITF translocation, an EZH2 mutation, a SUZ12 mutation,and/or an EED mutation.
 15. The method of claim 1, wherein effectiveamount decreases the activity of a BAF complex in the subject.
 16. Themethod of claim 1, wherein the effective amount inhibits BRM and/or BRG1in the subject.
 17. The method of claim 1, wherein the effective amountinduces apoptosis in the subject.
 18. The method of claim 1, wherein themetastatic uveal melanoma is resistant to, or failed to respond to,prior treatment with an anticancer therapy.
 19. The method of claim 1,wherein the method further comprises administering to the subject ananticancer therapy.
 20. The method of claim 19, wherein the anticancertherapy is surgery, a MEK inhibitor, and/or a PKC inhibitor, or acombination thereof.